Human Mesenchymal Progenitor Cell

ABSTRACT

The present invention provides isolated pluri-differentiated human mesenchymal progenitor cells (MPCs), which simultaneously express a plurality of genes that are markers for multiple cell lineages, wherein the multiple cell lineages comprise at least four different mesenchymal cell lineages (e.g., adipocyte, osteoblast, fibroblast, and muscle cell) and wherein each of the markers is specific for a single cell lineage. The present invention also method for isolating and purifying human mesenchymal progenitor cells from Dexter-type cultures for characterization of and uses, particularly therapeutic uses for such cells. Specifically, isolated MPCs can be used for diagnostic purposes, to enhance the engraftment of hematopoietic progenitor cells, enhance bone marrow transplantation, or aid in the treatment or prevention of graft versus host disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/887,582, filed Jul. 9, 2004, which claims the benefit of U.S.Provisional Patent Application No. 60/486,077, filed Jul. 9, 2003. U.S.patent application Ser. No. 10/887,582 is also a continuation-in-part ofU.S. patent application Ser. No. 10/263,419, filed Oct. 3, 2002, nowU.S. Pat. No. 7,049,072, which claims the benefit of U.S. ProvisionalPatent Application No. 60/327,140, filed Oct. 3, 2001, U.S. ProvisionalPatent Application No. 60/334,277, filed Nov. 28, 2001, U.S. ProvisionalPatent Application No. 60/352,636, filed Jan. 28, 2002, and U.S.Provisional Patent Application No. 60/412,450, filed Sep. 20, 2002. U.S.patent application Ser. No. 10/263,419 is also a continuation-in-part ofU.S. patent application Ser. No. 09/914,508, filed Nov. 7, 2001, nowU.S. Pat. No. 6,936,281, which is a National Stage Application ofInternational Application Number PCT/US01/16408, filed May 21, 2001,which claims the benefit of U.S. Provisional Patent Application No.60/277,700, filed Mar. 21, 2001, and U.S. Provisional Patent ApplicationNo. 60/209,245, filed Jun. 5, 2000. U.S. patent application Ser. No.10/887,582 is also a continuation-in-part of U.S. patent applicationSer. No. 09/914,508, filed Nov. 7, 2001, now U.S. Pat. No. 6,936,281.Each of the foregoing applications are incorporated herein by referencein their entirety, including all nucleic acid sequences, amino acidsequences, figures, tables, and claims.

GRANT INFORMATION

The subject matter of this application has been supported by a researchgrant from the National Heart Lung Blood Institute (NHLBI) and theNational Institutes of Health (NIH) under grant number HL59683.Accordingly, the government has certain rights in this invention.

Table 23 for this application is labeled “Table 23.txt”, was created onJul. 9, 2003, and is 1,124 KB. Table 24 for this application is labeled“Table 24.txt”, was created on Jul. 9, 2003, and is 4,716 kilobytes. Theentire contents of each of the tables are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention generally relates to pluri-differentiatedmesenchymal progenitor cells and therapeutic uses for the same. Morespecifically, the isolated mesenchymal progenitor cells are isolatedfrom hematopoietic cells and macrophages in Dexter-type cultures cells.

BACKGROUND OF THE INVENTION

Bone marrow, the site of blood cell production and home to variousleukemia and lymphoma cells, comprises a complex cellular populationincluding hematopoietic progenitor or stem cells and the stromal cellsthat support them. Hematopoietic stem cells have the capacity forself-regeneration and for generating all blood cell lineages whilestromal stem cells have the capacity for self-renewal and for producingthe hematopoietic microenvironment.

Two bone-marrow culture systems introduced in the mid-1970's haveevolved as favored media for the in vitro analysis of mesengenesis andhematopoiesis. The Friedenstein culture system was introduced in 1976 asa media for the analysis and study of mesengenesis (Friedenstein, et al,in Exp Hematol 4, 267-74 (1976)). It is necessary to first isolate rarepluripotant mesenchymal stem cells from other cells in the bone marrow.In the Friedenstein culture system, isolating the nonhematopoietic cellsis achieved by utilizing their tendency to adhere to plastic. Onceisolated, a monolayer of homogeneous, undifferentiated stromal cells isthen grown in the culture medium, in the absence of hematopoietic cells.The stromal cells from this system have the potential to differentiateinto discrete mesenchymal tissues, namely bone, cartilage, adiposetissue and muscle depending on specific growth supplements. These MSCshave been the target of extensive investigation including exploration oftheir potential clinical utility in repair or replacement of geneticallydamaged mesenchymal tissues.

In 1977, Dexter, et al. developed another bone marrow culture system forthe study of hematopoiesis (Dexter et al. J Cell Physiol 91, 335-44(1977)). The Dexter culture does not require isolation of themesenchymal cells before culturing. Thus, the monolayer of stromal cellsis grown in the presence of hematopoietic cells. Greenberger latermodified the Dexter system by the addition of hydrocortisone to theculture medium, making it more reproducible (Greenberger, Nature 275,752-4 (1978)).

Based on the Dexter system's ability to support sustained growth andpreservation of hematopoietic progenitor cells, it has become thestandard in vitro model for the study of hematopoiesis. Although theDexter-type stromal cells and the MSCs in Friedenstein-type culturesexpress similar cytokine/growth factor profiles, the Dexter cultureshave been found to be more efficient at maintaining preservation ofhematopoietic progenitor cells. Over the last 23 years, questions haveremained as to whether the cells from the Dexter cultures retained thepotential to differentiate, like the MSCs in the Friedenstein culture,or whether they have differentiated into another and discrete phenotypedue to their interaction with the hematopoietic cells (Prockop, Sciencev 276 n 5309, p 71 (4) (April 1997)). It has been widely believed thatthe stromal cells of the Dexter cultures are a heterogeneous mixture ofadipocytes, osteoblasts, fibroblasts, muscle cells, and vascularendothelial cells.

The in vitro analysis and study of hematopoiesis in Friedenstein andDexter culture systems has been of great importance in both veterinaryand human medicine. A number of diseases and immune disorders, as wellas malignancies, appear to be related to disruptions within thehematopoietic system.

Allogeneic bone marrow transplantation is the preferred treatment for avariety of malignant and genetic diseases of the blood and blood-formingcells. The success rate of allogeneic bone marrow transplantation is, inlarge part, dependent on the ability to closely match the majorhistocompatibility complex of the donor cells with that of the recipientcells to minimize the antigenic differences between the donor and therecipient, thereby reducing the frequency of host-versus-graft responsesand graft-versus-host disease (GvHD). Unfortunately, only about 20% ofall potential candidates for bone marrow transplantation have a suitablefamily member match.

Bone marrow transplantation can be offered to those patients who lack anappropriate sibling donor by using bone marrow from antigenicallymatched, genetically unrelated donors (identified through a nationalregistry), or by using bone marrow from a genetically related sibling orparent whose transplantation antigens differ by one to three of sixhuman leukocyte antigens from those of the patient. Unfortunately, thelikelihood of fatal GvHD and/or graft rejection increases from 20% formatched sibling donors to 50% in the cases of matched, unrelated donorsand unmatched donors from the patient's family.

The potential benefits of bone marrow transplantation have stimulatedresearch on the cause and prevention of GvHD. The removal of T cellsfrom the bone marrow obtained from matched unrelated or unmatchedsibling donors results in a decreased incidence of graft versus hostreactions, but an increased incidence of rejection of the allogeneicbone marrow graft by the patient.

Current therapy for GvHD is imperfect, and the disease can bedisfiguring and/or lethal. Thus, risk of GvHD restricts the use of bonemarrow transplantation to patients with otherwise fatal diseases, suchas severe immunodeficiency disorders, severe aplastic anemia, andmalignancies.

The potential to enhance engraftment of bone marrow or stem cells fromantigenically mismatched donors to patients without graft rejection orGvHD would greatly extend the availability of bone marrowtransplantation to those patients without an antigenically matchedsibling donor.

Thus, it would be useful to develop methods of improving and/or enhancebone marrow transplantation by enhancing the engraftment of bone marrowor hematopoietic progenitor cells and/or decreasing the occurrence ofgraft rejection or GvHD in allogenic transplants.

Studies of hematopoiesis and mesengenesis and the urgent need forimproved methods of treatment in the field of bone marrow transplantshave led to the isolation of MSCs from bone marrow stroma. These MSCsare the same pluri-potential cells that result from expansion inFriedenstein type cultures. Several patents describe the isolation andtherapeutic uses of these MSCs.

U.S. Pat. No. 5,486,359, to Caplan, et al., discloses isolated humanMSCs, and a method for their isolation, purification, and culturing.Caplan, et al. also describes methods for characterizing and using thepurified mesenchymal stem cells for research, diagnostic, andtherapeutic purposes. The invention in '359, to Caplan, et al.,describes pluri-potential cells that remain pluri-potential, even aftercultural expansion. Caplan, et al. also teaches that it is necessary tofirst isolate the pluri-potent MSCs from other cells in the bone marrowand then, in some applications, uses culture medium to expand thepopulation of the isolated MSCs. The Caplan et al. patent fails todisclose the use of Dexter-type cultures, pluri-differentiatedmesenchymal progenitor cells, or the isolation of cells from Dexter-typecultures.

U.S. Pat. No. 5,733,542, to Haynesworth, et al., discloses methods andpreparations for enhancing bone marrow engraftment in an individual byadministering culturally expanded MSC preparations and a bone marrowgraft. U.S. Pat. No. 6,010,696, to Caplan, et al., discloses methods andpreparations for enhancing hematopoietic progenitor cell engraftment inan individual by administering culturally expanded MSC preparations andhematopoietic progenitor cells. The cells utilized in the Haynesworth,et al. patent and the '696 patent to Caplan, et al. are thepluri-potential cells described in U.S. Pat. No. 5,486,359. Neitherpatent discloses the use of Dexter-type cultures, pluri-differentiatedmesenchymal progenitor cells, or the isolation of cells from Dexter-typecultures.

Mesenchymal stem cells that are isolated from bone marrow are furtherdescribed by Prockop, in Science v 276 n 5309, p 71 (4) (1997) andPittenger, et al. in Science v284 i5411, p 143 (1). These articles alsodescribe pluri-potential but undifferentiated MSCs and fail to teach ordisclose a pluri-differentiated mesenchymal cell or the isolation ofmesenchymal cells from Dexter-type cultures.

While the cells disclosed in the prior art may provide some benefit, theisolated MSCs in the prior art have not solved the problems associatedwith engraftment of hematopoietic progenitor cells or bone marrow.Consequently, there exists a need in the art for methods of improvingengraftment of hematopoietic progenitor cells and bone marrow in mammalsin need of such treatment. There also exists a need in the art fortreating and preventing the occurrence of GvHD in mammals that receiveallogeneic bone marrow transplants.

SUMMARY OF THE INVENTION

According to the present invention there is provided isolatedpluri-differentiated mesenchymal progenitor cells, a method ofisolation, diagnostic uses, and therapeutic uses relating to enhancingthe engraftment of human bone marrow or hematopoietic progenitor cellsand treating GvHD.

The present invention provides an isolated mesenchymal progenitor cellthat is pluri-differentiated.

Accordingly, the present invention also provides a method for purifyingpluri-differentiated mesenchymal progenitor cells including the stepsof: providing a cell culture preparation by the Dexter method, treatingthe cells to obtain a cell suspension, removing macrophages,fractionating the cells, and collecting the fraction ofpluri-differentiated mesenchymal progenitor cells.

The present invention also provides a method for enhancing bone marrowengraftment in a mammal in need thereof which includes administering tothe mammal (i) isolated pluri-differentiated mesenchymal progenitorcells and (ii) a bone marrow graft, wherein the pluri-differentiatedmesenchymal progenitor cells are administered in an amount effective topromote engraftment of the bone marrow in the mammal.

The present invention provides a method for enhancing engraftment ofhematopoietic progenitor cells in a mammal in need thereof whichincludes the step of administering to the mammal (i) isolatedpluri-differentiated mesenchymal progenitor cells and (ii) hematopoieticprogenitor cells, wherein the pluri-differentiated mesenchymalprogenitor cells are administered in an amount effective to promoteengraftment of the hematopoietic progenitor cells in the mammal.

Another embodiment of the present invention provides a method fortreating graft-versus-host disease (GvHD) in a mammal about to undergobone marrow or organ transplantation or suffering from GvHD caused bybone marrow or organ transplantation, by administering to the mammal aneffective amount of isolated pluri-differentiated mesenchymal progenitorcells.

Yet another embodiment of the present invention provides a method fordiagnosing a disease state by: a) establishing gene expression patternsof normal state bone marrow derived isolated pluri-differentiatedmesenchymal progenitor cells; b) establishing gene expression patternsof various leukemic state bone marrow derived isolatedpluri-differentiated mesenchymal progenitor cells; c) identifying genesets that are unique to a given state; and d) comparing a profile ofbone marrow derived isolated mesenchymal progenitor cell of unknownstate to the gene sets.

Additionally, the present invention provides a method for identifyingtherapeutic targets for treatment of hematopoietic function by: a)determining the median gene expression profile of bone marrow isolatedpluri-differentiated mesenchymal progenitor cells associated with eachdisease state of interest; b) identifying gene groups that areup-regulated, down regulated, and common to each disease state; and c)identifying gene sets that are unique to a given state.

The present invention also includes therapeutic compositions includingisolated pluri-differentiated mesenchymal progenitor cells and apharmaceutically acceptable carrier, wherein the pluri-differentiatedmesenchymal progenitor cells are present in an amount effective toenhance bone marrow engraftment in a mammal in need thereof, enhancehematopoietic progenitor cell engraftment in a mammal in need thereof;or treat GvHD in a mammal about to undergo bone marrow or organtransplantation or suffering from GvHD caused by bone marrow or organtransplantation.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Other advantages of the present invention can be readily appreciated asthe same becomes better understood by reference to the followingdetailed description when considered in connection with the accompanyingdrawings. The following is a brief description of the drawings which arepresented only for the purposes of further illustrating the inventionand not for the purposes of limiting same. Referring to the drawingfigures, like reference numerals designate identical or correspondingelements throughout the several figures.

FIG. 1 is a photograph showing the phase contrast photomicrograph viewof a Dexter-type stromal cell monolayer reflecting on cellularcomplexity.

FIG. 2 is a photograph showing the percoll gradient centrifugationtechnique of the present invention that purifies the MPCs (2) in largequantities to greater than 95% purity.

FIG. 3 is a photograph showing the Wright-Giemsa staining of Dexter-typestromal cell cultures depicting three morphologically identifiable cellpopulations, macrophages (5), hematopoietic cells (3), and themesenchymal progenitor cells (4) of the present invention.

FIGS. 4A-H show a series of photomicrographs showing the morphologic andphenotypic characteristics of the MPCs of the present invention, asuncovered by staining for representative mesenchymal cell lineagemarkers. The methods applied are shown in parentheses. (FIG. 4A)Wright-Giemsa (Harleco stain using HMS Series Programmable SlideStainer, Carl Zeiss, Inc.). (FIG. 4B) Immunostain using anti-CD68antibody (Immunotech, Clone PG-M1; Vector, Vectastain Elite ABC Kit).(FIG. 4C) Immunostain using anti-CD45 antibody (Dako, Clone PD7/26 &2B11; ABC Kit). (FIG. 4D) Periodic acid-Schiff (Sigma). (FIG. 4E) NileRed (Sigma), counterstained with DAPI (Vector). (FIG. 4F) Alkalinephosphatase (Sigma Kit No. 85), counterstained with Nuclear Fast Red(Baker). (FIG. 4G) Immunostain using antibody to fibronectin(Immunotech, Clone 120.5; ABC Kit). (FIG. 4H) Immunostain usinganti-muscle actin antibody (Ventana, clone HUC 1-1; Ventana system usinga section of formalin-fixed, paraffin-embedded cell block, instead of acytospin). Appropriate positive controls and isotype-matched negativecontrols were employed to ascertain antibody staining-specificity. Allparts of figure as shown, except 4E and 4H, have clearly identifiablebuilt-in cell controls. The morphological features of the cells arelisted in row 1 of Table 1.

FIG. 5 is a photograph which shows a transmission electron micrograph ofan MPC of the present invention bearing microvilli, irregular nucleus,and pools of glycogen (6) in the ectoplasm (×4,600).

FIGS. 6A-M are photographs which show Northern blot analysis of bonemarrow stromal cell RNAs for expression of genes specific for multiplemesenchymal cell lineages. FIGS. 6A-M represent different gene probesused for hybridization. The following outlines the sources of the geneprobes employed and the approximate sizes of the major transcriptsobserved (shown in parentheses): FIG. 6A) CD68 (Clone ID 3176179, GenomeSystems, Inc (GSI); 2-3 kb); FIG. 6B) Cathepsin B (Clone ID 2806166,GSI; 2-3 kb); FIG. 6C) GAPDH probe (generated using PCR primers from R&DSystems, Inc; ˜2 kb) hybridized to same blot as A and B; FIG. 6D)Adipsin (probe generated using PCR primers as described, Ref 20; 0.5-1kb); FIG. 6E) Osteoblast-specific cadherin-11 (Clone ID 434771, GSI; 3kb); FIG. 6F) Chondroitin sulfate proteoglycan 2 (Clone ID 1623237,GSI; >10 kb); FIG. 6G) Collagen type I alpha 1 (Clone ID 782235,GSI; >10 kb); FIG. 6H) Decorin (Clone ID 3820761, GSI; 2-3 kb); FIG. 6I)GAPDH probe hybridized to same blot as D-H; FIG. 6J) Fibronectin (CloneID 3553729, GSI; >10 kb); FIG. 6K) Caldesmon (Clone ID 1319608, GSI; 4kb); FIG. 6L) Transgelin (Clone ID 4049957, GST; ˜1.5 kb); and FIG. 6M)GAPDH probe hybridized to same blot as J-L.

FIG. 7 is a photograph which shows RT-PCR analysis for expression ofrepresentative hematopoietic growth factors (G-CSF and SCF) andextracellular matrix receptors (ICAM-1, VCAM-1, and ALCAM) by the MPCsof the present invention.

FIG. 8 is a graph comparing of the ability to support in vitrohematopoiesis by the purified MPCs (heavy fraction represented by gray)of the present invention vs. unfractionated bone marrow stromal cells(represented by black).

FIGS. 9A and 9B are graphs showing flow cytometric evidence of humanhematopoietic cell engraftment in a SCID mouse cotransplanted with theMPCs of the present invention. FIG. 9A shows CD45+/CD34+ progenitors inthe marrow. FIG. 9B shows CD45/CD34-mature hematopoietic cellscirculating in the blood.

FIGS. 10A-H are photographs which show engraftment of humanhematopoietic cells in a SCID mouse cotransplanted with the purifiedmarrow MPCs of the present invention. FIG. 10A shows a serial section ofa mouse spleen stained with H & E. FIG. 10B shows a serial section of amouse spleen stained with immunoperoxidase stain for CD45. FIG. 10Cshows bone marrow stained for CD45. FIG. 10D shows a serial section ofthe mouse liver stained with H&E depicting involvement of periportalareas. FIG. 10E shows a serial section of the mouse stomach stained withH&E showing transmural infiltration. FIG. 10F shows a serial section ofthe mouse lung stained with H&E showing involvement of peribronchialarea. FIG. 10G shows a serial section of the mouse pancreas stained withH&E. FIG. 10H shows a serial section of the mouse paravertebral gangliastained with H&E.

FIG. 11A-C is a photomicrograph of a serial section of the spleen of anormal BALB/C mouse showing white pulp populated by darkly staininglymphocytes (H&E). FIG. 11B is a photomicrograph of the spleen of a SCIDmouse showing white pulp largely consisting of lightly staining stromalframework (H&E). FIG. 11C is a photomicrograph of the spleen of a SCIDmouse cotransplanted with human bone marrow MNC and the purified bonemarrow MPCs of the present invention showing homing (engraftment) ofhuman B cells to white pulp.

FIGS. 12A-C are photographs which show Southern blotting data. FIG. 12Ashows that hybridization of sample DNA using a DNA probe specific forhuman chromosome 17 alpha satellite DNA (p17H8) results in a 2.7 Kb band(7) (arrow; autoradiogram exposed for only 45 minutes). FIG. 12B showsEcoR1 digest of thymic genomic DNA from SCID mice. FIG. 12C shows EcoR1digest of lymph node genomic DNA from SCID mice.

FIGS. 13A-1, 13A-2, 13B-1, and 13B-2 show graphs comparing the survivalrate and engraftment of human hematopoietic cells in SCID micecotransplanted with the purified bone marrow MPCs of the presentinvention vs. unpurified bone marrow stromal cells. In the line graphsprovided the line with diamonds represents MPCs and bone marrowmononuclear cells, squares represents bone marrow mononuclear cellsonly, triangles represents unfractionated bone marrow stromal cells, theXs represent MPCs only, and the circles represent the control. In thebar graphs, the gray bars represent mice that survived and the blackbars represent mice with engraftment.

FIGS. 14A-D are photographs which demonstrate apoptosis by TUNEL assayin organs of SCID mice that died after transplantation. FIG. 14A shows aserial section of the liver of the mouse that survived. FIG. 14B shows aserial section of the liver of the mouse that died. FIG. 14C shows aserial section of the spleen of the mouse that survived. FIG. 14D showsa serial section of the spleen of the mouse that died.

FIG. 15 shows photomicrographs of single-cell MPCs that were isolated bylaser capture microdissection (LCM) and subsequently targeted formicroarray analysis.

FIG. 16 shows a Venn diagram displaying the stromal-cell gene-list.Stromal cell genes are operationally defined as being active in at least9 out of 10 single cell MPCs AND 4 out of 5 collective MPC samples AND 7out of 8 collective USC samples, i.e., 20 of 23 samples tested. Thiscriterion was very stringent and automatically excluded the outliers,independently of filtering for genes with weak expressions on the basisof control strength (referred to as C or CS). The stromal cell gene listof 2755 includes 13 AFFX microarray-assay positive controls.

FIG. 17 shows a two-dimensional hierarchical clustering of 2755 stromalcell genes based on the expression profiles of 23 samples. The gene treeis displayed on top and the experiment or sample tree is shown on left.Accordingly, each column represents a particular gene on the chip andeach row represents a separate stromal cell sample.

FIG. 18 shows composite gene-expression plots of 2755 stromal cell genescomparing collective purified stromal cell samples (cMPC), collectiveunpurified stromal cell samples (cUSC) and single-cell stromal cellsamples (sMPC). Individual samples are represented on X-axis. Normalizedintensity of gene expression is shown on Y-axis in log scale.

FIGS. 19A-19F show gene-expression plots of diverse mesenchymallineage-associated genes and housekeeping genes by collective MPCs andsingle-cell MPCs. Individual samples are represented on X-axis. Signalintensity of a transcript in log scale normalized across samples isshown on Y-axis. Note the differing log scales, particularly the widerange of log scale for ACTB. Representative lineage markers are shown asfollows. Osteoblast markers: osteoblast-specific factor 2 (probe ID1451_s-at), osteoblast cadherin 11 (ID 2087_s_at) and collagen 1 alpha 2(ID 32306_g_at). Muscle markers: caldesmon (ID 41738_at), transgelin-2(ID 36678_at) and smooth muscle myosin heavy chain (ID 32838_at).Fibroblast markers: fibronectin (ID 31719_at) and prolyl 4-hydroxylase(ID 37037_at). Adipocyte markers: adipsin (ID 40282_at) andadipocyte-specific ECM protein (ID 39673_i_at). Housekeeping genes: GAPD(ID 35905_s_at) and ACTB (ID 32318_s_at). Samples 1-5, respectively,represent MPC A, MPC B R2, MPC C R2, MPC D R1, MPC D R2. Samples 6-15,respectively, represent SCA1, SCA2, SCA3, SCB1, SCB3, SCC1, SCC3, SCD1,SCD2, SCD3.

FIG. 20A-20F shows gene-expression plots of representative precursorB-lymphocyte-associated genes by collective MPCs and single-cell MPCs.Individual samples are represented on X-axis. Signal intensity of atranscript in log scale normalized across samples is shown on Y-axis.Note that the CD markers that are traditionally associated withhematopoietic cells, CD45 (probe ID 40518_at), CD19 (ID 1116_at) andCD34 (ID (538_at), are expressed by sMPCs. CD45, when present, is moreabundantly detected in single MPCs than in collective MPCs, and isparticularly noticeable by wide range of log scale for CD45. The otherpre-B cell associated markers that are expressed by sMPCs are CD10 (ID1389_at), HLA-Dr (ID 33261_at) and CD79A (ID 34391_at). Samples 1-5,respectively, represent MPC A, MPC B R2, MPC C R2, MPC D R1, MPC D R2.Samples 6-15, respectively, represent SCA1, SCA2, SCA3, SCB1, SCB3,SCC1, SCC3, SCD1, SCD2, SCD3.

FIGS. 21A-21F show gene-expression plots of representative precursorB-lymphocyte-associated genes by collective MPCs and single-cell MPCs.Individual samples are represented on X-axis. Signal intensity of atranscript in log scale (normalized across 15 samples) is shown onY-axis. The CD markers that are traditionally associated withhematopoietic cells, CD45 (probe ID 40518_at), CD19 (ID 1116_at) andCD34 (ID (538_at), are expressed by sMPCs. CD45, when present, is moreabundantly detected in single MPCs than in collective MPCs, and isparticularly noticeable by wide range of log scale for CD45. The otherpre-B cell associated markers that are expressed by sMPCs are CD10 (ID1389_at), HLA-Dr (ID 33261_at) and CD79A (ID 34391_at). Samples 1-5,respectively, represent MPC A, MPC B R2, MPC C R2, MPC D R1, MPC D R2.Samples 6-15, respectively, represent SCA1, SCA2, SCA3, SCB1, SCB3,SCC1, SCC3, SCD1, SCD2, and SCD3.

FIGS. 22A-22F show scatter plots using log transformed data and showingsystematic analysis of transcriptome wide random variation. The methodsinvolved in construction of scatter plots are described in the sectionentitled, “Second-tier data-analysis/data mining”. The results arediscussed in the section entitled “Data mining and reproducibility ofoverall procedures”.

DETAILED DESCRIPTION OF THE INVENTION

Generally, the present invention provides isolated and purifiedmesenchymal progenitor cells that are pluri-differentiated. Alsoprovided by the present invention is a therapeutic (pharmaceutical)composition including an effective amount of isolated and purifiedpluri-differentiated mesenchymal progenitor cells and a pharmaceuticallyacceptable carrier.

In one aspect, the present invention provides an isolatedpluri-differentiated mesenchymal progenitor cell, wherein the cellsimultaneously expresses a plurality of genes that are markers formultiple cell lineages, wherein the multiple cell lineages comprise atleast four different mesenchymal cell lineages, and wherein each of themarkers is specific for a single cell lineage. The terms “mesenchymalprogenitor cell”, “MPC”, and “pluri-differentiated mesenchymalprogenitor cell” are used interchangeably herein to refer to theaforementioned cells of the subject invention.

In one embodiment, the MPC is not a cell of a cell line. In anotherembodiment, the at least four different mesenchymal cell lineagescomprise adipocyte, osteoblast, fibroblast, and muscle cell. In anotherembodiment, the markers are specific for a single cell lineage areselected from the group consisting of Nile Red, Oil Red O, adipsin,alkaline phosphatase, cadherin-11, chondroitin sulfate, collagen type I,decorin, fibronectin, prolyl-4-hydroxylase, actin, caldesmon, andtransgelin. In another embodiment, the MPC simultaneously expresses theplurality of genes in the presence of hydrocortisone and horse serum.Preferably, the MPC is not a neoplastic cell, and is chromosomallynormal, as determined by Geimsa-trypsin-Wrights (GTW) banding. In oneembodiment, the cell is a human cell. The MPC is obtainable directlyfrom a primary cell culture. Preferably, the primary culture is a Dexterculture. In another embodiment, the MPC is not immortalized.

In another embodiment, the MPC is obtained by providing a cell culturepreparation by the Dexter method, treating the cells of the cell culturepreparation to obtain a cell suspension, removing macrophages from thecell suspension, fractionating the remaining cells, and collecting thefraction of cells containing the isolated cell. The fractionating stepmay involve any suitable cell separation technique known in the art,such as fractionation based on density gradient (e.g., Percollgradient), use of ferromagnetic beads, cytometry, and fluorescenceactivated cell sorting.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising isolated MPCs and a pharmaceutically acceptablecarrier. Preferably, the MPCs are present in an amount effective fortreating a disease state in a mammal in need thereof. In one embodiment,the MPC are present in an amount effective to enhance hematopoieticprogenitor cell engraftment in a mammal in need thereof. Preferably, thecarrier is sterile, such as sterile saline. In another embodiment, theMPC are present in an amount effective to treat graft-versus-hostdisease (GvHD) in a mammal about to undergo bone marrow or organtransplantation or suffering from GvHD caused by bone marrow or organtransplantation. Optionally, the composition further comprises cellsother than MPCs, or tissue, for transplantation. In one embodiment, thetissue comprises bone marrow. In another embodiment, the tissuecomprises an organ.

In one embodiment, the MPC of the pharmaceutical composition of theinvention are obtained by providing a cell culture prepared by theDexter method, treating the cells of the cell culture to obtain a cellsuspension, removing macrophages from the cell suspension, fractionatingthe remaining cells, and collecting the fraction containing thepluri-differentiated mesenchymal progenitor cells.

In another aspect, the present invention provides a plurality ofisolated MPC (i.e., pluri-differentiated mesenchymal progenitor cells,wherein the plurality of cells are cells that individuallysimultaneously express a plurality of genes that are markers formultiple cell lineages, wherein the multiple cell lineages comprise atleast four different mesenchymal cell lineages, and wherein each of themarkers is specific for a single cell lineage). Preferably, the MPCshave been isolated from hematopoietic cells and macrophages to a purityof at least 95%.

In one embodiment, the plurality of isolated MPC are obtained byproviding a cell culture preparation by the Dexter method, treating thecells of the cell culture preparation to obtain a cell suspension,removing macrophages from the cell suspension, fractionating theremaining cells, and collecting the fraction of cells containing theplurality of cells.

In another aspect, the present invention provides a method for purifyingpluri-differentiated mesenchymal progenitor cells comprising the stepsof: (a) providing a cell culture preparation by the Dexter method; (b)treating the cells to obtain a cell suspension; (c) removing macrophagesfrom the cell suspension; (d) fractionating the remaining cells; and (e)collecting the fraction of pluli-differentiated mesenchymal progenitorcells, wherein the pluri-differentiated mesenchymal progenitor cellsindividually simultaneously express a plurality of genes that aremarkers for multiple cell lineages, wherein the multiple cell lineagescomprise at least four different mesenchymal cell lineages, and whereineach of the markers is specific for a single cell lineage.

In another aspect, the present invention provides a method for enhancingengraftment of cells in a human or non-human mammal in need thereof, themethod comprising administering to the mammal isolatedpluri-differentiated mesenchymal progenitor cells of the invention,wherein the isolated pluri-differentiated mesenchymal progenitor cellsare administered in an amount effective to promote engraftment of thecells. In one embodiment, the isolated pluri-differentiated mesenchymalprogenitor cells are administered by intravenous injection or byinjecting directly to the site of intended activity. Optionally, themethod further comprises administering the cells for engraftment,wherein the cells are administered before, during, or after the isolatedpluri-differentiated mesenchymal progenitor cells are administered. Inone embodiment, the cells to be engrafted comprise hematopoieticprogenitor cells. In another embodiment, the isolatedpluri-differentiated mesenchymal progenitor cells are administered tothe mammal in a cell suspension further comprising hematopoieticprogenitor cells.

In another aspect, the present invention provides a method for enhancingbone marrow engraftment in a mammal in need thereof, the methodcomprising administering to the mammal (i) isolated pluri-differentiatedmesenchymal progenitor cells of the invention and (ii) a bone marrowgraft, wherein the isolated pluri-differentiated mesenchymal progenitorcells are administered in an amount effective to promote engraftment ofthe bone marrow in the mammal. Advantageously, the isolatedpluri-differentiated mesenchymal progenitor cells are capable ofincreasing the survival of hematopoeitic cells transplantedsimultaneously or consecutively. In one embodiment, the administeringcomprises intravenously injecting or directly injecting the isolatedpluri-differentiated mesenchymal progenitor cells to the site ofintended engraftment.

In another aspect, the present invention provides a diagnostic methodfor screening isolated pluri-differentiated mesenchymal progenitor cellsfor abnormalities, comprising isolating RNA from the isolatedpluri-differentiated mesenchymal progenitor cells for abnormalities;amplifying the isolated RNA; analyzing the amplified RNA using nucleicacid array (e.g., microarray); determining one or more gene expressionpatterns; and comparing the determined one or more gene expressionpatterns to one or more gene expression patterns of normalpluri-differentiated mesenchymal progenitor cells. The diagnostic methodmay be used to screen for a hematologic disease or other diseaseseffecting stromal cells, for example. In one embodiment, theabnormalities are phenotypic abnormalities that can be discerned at asingle cell level.

Amplification of nucleic acids is typically performed prior to arrayingthe nucleic acids. Commonly, amplification involves one or more nucleicacid amplifications, e.g., by a PCR, TMA, NASBA or RCA reaction.Optionally, the PCR is an rtPCR that couples reverse transcription andamplification of the expressed RNA samples. The amplification can beeither a global amplification or a selective (e.g., target specific)amplification of one or more species in the expressed RNA sample(s).Each expressed RNA sample can be amplified in two or more targetspecific amplification arrays, and, for example, spatially arrayed intwo or more locations on a physical array. Optionally, a plurality ofdefined sequence probes each of which specifically hybridizes to theproducts of a different target specific amplification reaction ishybridized to the array. In some embodiments, amplification products arepooled for arraying.

A variety of nucleic acid array formats can be employed in the contextof the present invention. In some embodiments, the arrays are solidphase arrays, i.e., the nucleic acids are arrayed on one or more solidphase surface. In some embodiments, the nucleic acids corresponding toexpressed RNA samples are arrayed on a two dimensional solid phasesurface. In alternative embodiments, the nucleic acids are arrayed on aplurality of solid phase surfaces, such as beads, spheres, pins, oroptical fibers. Solid phase arrays surfaces can include a variety ofmaterials, and in various embodiments of the invention, the arraysurface is composed, e.g., of glass, coated glass, silicon, poroussilicon, nylon, ceramic or plastic. In various embodiments of theinvention, expressed RNA samples for analysis are obtained from avariety of biological sources or samples (e.g., bone marrow derivedcultures) which have been exposed to or treated with members of alibrary of compositions (such as cytokines) or agents of potentialtherapeutic value.

In another aspect, the invention provides a method for reducinggraft-versus-host disease (GvHD) in a mammal caused by bone marrow ororgan transplantation, the method comprising administering to the mammalan effective amount of isolated pluri-differentiated mesenchymalprogenitor cells of the present invention.

In another aspect, the invention provides a method for diagnosing adisease state comprising the steps of: (a) establishing gene expressionpatterns of normal state bone marrow derived isolatedpluri-differentiated mesenchymal progenitor cells; (b) establishing agene expression pattern for bone marrow derived isolatedpluri-differentiated mesenchymal progenitor cells of different leukemicstates; (c) identifying gene sets that are unique to a given leukemicstate; and (d) comparing a profile of a bone marrow derived isolatedmesenchymal progenitor cell of unknown state to the gene sets.

In another aspect, the invention provide a method for diagnosing adisease state in a patient, the method comprising: (a) providing a geneexpression profile of a bone marrow derived isolatedpluri-differentiated mesenchymal progenitor cell of unknown state fromthe patient; and (b) comparing the patient gene expression profile to atleast one reference gene expression profile to diagnose a disease statein the patient, wherein the reference gene expression profile is a geneexpression profile of a bone marrow derived isolatedpluri-differentiated mesenchymal progenitor cell in a leukemic state orin a normal state. In one embodiment, the comparing step comprisescomparing the patient gene expression profile to a plurality ofreference gene expression profiles, wherein each of the reference geneexpression profiles is associated with a different leukemic state. Eachreference gene expression profile can comprise genes differentiallyexpressed in the leukemic state compared to the normal state.

In one embodiment, the differentially expressed genes comprise at leastone class of genes selected from the group consisting of annexins,caspases, cadherins, calmodulins, calmodulin-dependent kinases, celladhesion molecules, cathespins, collagens, cytokines, epidermal growthfactors, fibroblast growth factors, fibronectins, galectins, growthfactors, genes of the IGF system, interleukins, interleukin receptors,integrins, disintegrins, lineage-specific markers, laminins,platelet-derived growth factors, platelet-derived growth factorreceptors, interferon-gamma, TNF-alpha, and TGF-beta. In a specificembodiment, the differentially expressed genes comprise TNF-alpha,TGF-beta, and interferon-gamma.

Each reference gene expression profile can comprise expression values ofgenes differentially expressed in the leukemic state compared to thenormal state. In one embodiment, the at least one reference geneexpression profile is contained within a database. Preferably, thecomparing step is carried out using a computer algorithm. Optionally,the method further comprises (c) selecting the reference gene expressionprofile most similar to the patient gene expression profile, to diagnosethe patient. Optionally, the method further comprises isolating the bonemarrow derived isolated pluri-differentiated mesenchymal progenitor cellof unknown state from the patient.

In one embodiment, the at least one reference gene expression profilecomprises a gene expression profile of a bone marrow derived isolatedpluri-differentiated mesenchymal progenitor cell in a leukemic state anda gene expression profile of a bone marrow derived isolatedpluri-differentiated mesenchymal progenitor cell in a normal state.

Optionally, the method further comprises preparing the patient geneexpression profile. The at least one reference gene expression profilecan be prepared by cluster analysis, for example.

In another embodiment, the method further comprises: (c) providing agene expression profile of a bone marrow derived isolatedpluri-differentiated mesenchymal progenitor cell from the patient afterthe patient has undergone a treatment regimen for a leukemic diseasestate; and (d) comparing the post-treatment patient gene expressionprofile to the at least one reference gene expression profile, tomonitor the patient's response to the treatment regimen.

The leukemic state may be a preleukemic condition, such asmyelodysplastic syndrome (MDS). The leukemic can be an over leukemia.The leukemic state can be a lymphoma, for example. In one embodiment,the leukemic state is selected from the group consisting of acutemyeloid leukemia (AML), chronic myeloid leukemia (CML), acutelymphoblastic leukemia (ALL), chronic lymphocyte leukemia (CLL), andmultiple myeloma (MM).

Optionally, the method may further comprise (c) providing a diagnosis ofthe disease state to the patient.

In one embodiment, the bone marrow derived isolated pluri-differentiatedmesenchymal progenitor cell of unknown state comprises a single cell.

In another embodiment, the bone marrow derived isolatedpluri-differentiated mesenchymal progenitor cell of unknown statecomprises a plurality of cells.

In one embodiment, the isolated pluri-differentiated mesenchymalprogenitor cells have been obtained by providing a cell culturepreparation by the Dexter method, treating the cells of the cell culturepreparation to obtain a cell suspension, removing macrophages from thecell suspension, fractionating the remaining cells, and collecting thefraction of cells containing the normal state pluri-differentiatedmesenchymal progenitor cells.

Typically, isolated pluri-differentiated mesenchymal progenitor cellsindividually share the characteristic of simultaneously expressing aplurality of genes that are markers for multiple cell lineages, whereinthe multiple cell lineages comprise at least four different mesenchymalcell lineages, wherein each of the markers is specific for a single celllineage, and wherein the cells are not cells of a cell line.

In another aspect, the present invention provides a method foridentifying therapeutic targets for treatment of hematopoietic functioncomprising the steps of: (a) determining the median gene expressionprofile of isolated pluri-differentiated mesenchymal progenitor cellsassociated with each disease state of interest; (b) identifying genegroups that are up-regulated, down regulated, and common to each diseasestate; and (c) identifying gene sets that are unique to a given diseasestate.

The terms “enhance” or “improve” as used herein are intended to indicatethat the there is a more beneficial end result. In other words, theproduct provides a more effective result.

In another aspect, the present invention provides a method of selectinga therapy for a patient, the method comprising: a) providing a subjectexpression profile of a pluri-differentiated mesenchymal progenitor cellfrom the patient; b) providing a plurality of reference gene expressionprofiles, each associated with a therapy, wherein the subject expressionprofile and each reference profile has a plurality of values, each valuerepresenting the expression level of a gene disclosed herein as beingexpressed in pluri-differentiated mesenchymal progenitor cells; and c)selecting the reference profile most similar to the subject expressionprofile, to thereby select a therapy for said patient. Optionally, themethod further comprises administering the therapy selected in step c)to the patient. In one embodiment, the most similar reference profile isselected by weighting a comparison value for each value of the pluralityusing a weight value associated with the particular gene. In oneembodiment, the subject expression profile has at least twenty values.

The term “pluri-differentiated” as used herein refers to cells that area single cell type co-expressing genes specific for multiple lineages.The term “pluri-potential” as used herein refers to cells that areundifferentiated and have the potential to be differentiated intodiscrete mesenchymal tissues.

Dexter-type cultures contain stromal cells that co-express multiplemessage lineage markers. These pluri-differentiated cells are referredto by the inventor as mesenchymal progenitor cells (MPCs). Disclosedherein is a process for isolating and purifying MPCs from Dexter-typecultures. Purified MPCs provide a sufficiently defined system to permitdetailed elucidation of the role of bone marrow in normal and leukemichematopoiesis.

The present invention also provides various methods for using MPCs toenhance bone marrow transplantation, enhance hematopoietic progenitorcell engraftment, for diagnostic purposes, or for the treatment of GvHD.

The exact cell types in Dexter cultures have now been identified. Noevidence was found for the existence of discrete cellular populations,such as adipocytes, osteoblasts, fibroblasts, smooth muscle cells andendothelial cells, notwithstanding the abundance of literature and widespread belief (See, J. L. Liesveld et al., Blood 73, 1794 (1989); A. K.Sullivan, D. Claxton, G. Shematek et al., Lab Invest 60, 667 (1989); K.Dorshlind, Ann Rev Immunol 8, 126 (1990); S. Perkins, R. A. Fleischman,Blood 75, 620 (1990); I. A. Denkers, R. H. Beelen, G. J. Ossenkoppele etal., Ann Hematol 64, 210 (1992); P. E. Penn, D. Z. Jiang, R. G. Fei etal., Blood 81, 1205 (1993); E. de Wynter et al., J Cell Sci 106, 761(1993); A. Ferrajoli et al., Stem Cells (Dayt) 12, 638 (1994); B. R.Clark, A. Keating, Ann NY Acad Sci 770, 70 (1995); B. S. Wilkins, D. B.Jones, Br J Haematol 90, 757 (1995); S. Gronthos, P. J. Simmons, JHematother 5, 15 (1996); D. Soligo et al., Abstract #3926, Blood 94,Supplement 1 (Part 2 of 2), p. 168b, Forty 1^(st) Annual Meeting of theAmerican Society of Hematology, New Orleans, La., Dec. 3-7, 1999, M-A.Dorheim et al., J Cell Physiol 154, 317 (1993), M. K. Majumdar, M. A.Thiede, J. D. Mosca et al., J Cell Physiol. 176, 57 (1998), D. J.Prockop, Science 276, 71 (1997), R. S. Taichman, S. G. Emerson, J ExpMed 179, 1677 (1994); R. S. Taichman, M. J. Reilly, S. G. Emerson, Blood87, 518 (1996); C. M. Verfaillie, in HEMATOLOGY: Basic Principles andPractice, R. Hoffman, et al., Eds. (Churchill Livingstone, N.Y., 2000),pp. 140-142.), A. J. Henderson, A. Johnson, K. Dorshkind, J Immunol 145,423 (1990); M. W. Long, J. L. Williams, K. G. Mann, J Clin Invest 86,1387 (1990); P. J. Simmons, S. Gronthos, A. Zannettino et al., Prog ClinBiol Res 389, 271 1994); B. A. Roecklein, B. Torok-Storb, Blood 85, 997(1995); J. Wineman, K. Moore, I. Lemischka et al., Blood 87, 4082(1996); K. A. Kelly, J. M. Gimble, Endocrinology 139, 2622 (1998); K. C.Hicok et al., J Bone Miner Res 13, 205 (1998); S. R. Park, R. O. Oreffo,J. T. Triffitt, Bone 24, 549 (1999); J. E. Dennis et al., J Bone MinerRes 14, 700 (1999); and B. Torok-Storb et al., Ann NY Acad Sci 872, 164(1999)). Instead, the inventor determined that there are only threetypes of cells in Dexter-type cultures, namely, macrophages (˜35%),hematopoietic cells (˜5%), and a type applicant calls “nonhematopoieticcells” (˜60%) (FIG. 3, FIG. 4A, and Table 1).

Bone marrow mesenchymal cells, the nonhematopoietic cells in Dexter typecultures, possess distinctive features that have previously goneunrecognized. There is both direct visual (FIGS. 4A-E and FIG. 5) andmolecular biological (FIG. 6) evidence to support the existence of thisunique cell type. These findings challenge the prevailing belief thatstromal cells derived from Dexter cultures comprise multiplesingly-differentiated mesenchymal cell types. Because Dexter culturesrepresent a primary cell culture system, and not a cell line, thesestudies indicate that cells in these primary cultures themselves arepluri-differentiated, which has been previously unsuspected. Thenonhematopoietic cells of the present invention (MPCs) simultaneouslyexpress marker genes specific for multiple mesenchymal cell lineages,including adipocytes, osteoblasts, fibroblasts and smooth muscle cells.As shown in the present disclosure, MPCs can also differentiate into Bcells and therefore be useful in affecting the functionality of theimmune system.

The MPCs in Dexter type cultures were characterized using a variety oftechniques. Cytospins were prepared using aliquots of unfractionatedcells for performance of various cytological, cytochemical andimmunocytochemical stains. Reactivity patterns of the bone marrowculture cells are outlined in Table 1. FIGS. 4A-E illustrate morphologicand phenotypic characteristics, as uncovered by staining forrepresentative cell lineage markers.

Only rarely have investigators in this field taken the approach ofpreparing a cell suspension and staining cells on cytospins as was doneto characterize the cells of the present invention (Simmons, et al.,Nature 328, p 429-32 (1987)) and no other group has used this method toaddress the issue of pluri-differentiation by bone marrow stromal cells.Almost all of the published studies in the field, with a rare exception(Simmons, et al., Nature, 328, p 429-32 (1987)), conducted cytochemicaland immunocytochemical staining on layers of stromal cells grown toconfluence on coverslips. In this situation, the stromal cultures appearvery complex especially in the areas of hematopoietic activity,so-called “cobblestones” with macrophages and hematopoietic cellsenmeshed in them. Macrophages and nonhematopoietic cells spreadthemselves and assume varied shapes when they adhere to and grow onplastic or glass. This spreading further contributes to the perceivedheterogeneity and complexity. The complexity precludes a clearmorphological visualization of the nonhematopoietic cells andconsequently interferes with the determination of what percent of whatcell type is positive for any given marker.

In terms of lineage markers, up to 100% of the nonhematopoietic cells orMPCs of the present invention expressed two fat cell markers (Nile Red(FIG. 4E) and Oil Red O); an osteoblast marker (alkaline phosphatase(FIG. 4F)); and two fibroblast markers (fibronectin (FIG. 4G) andprolyl-4-hydroxylase). Greater than 85% of the MPCs were also positivefor a muscle marker, actin (FIG. 4H). There was no evidence ofexpression of endothelial cell differentiation, as judged byimmunohistochemical staining for CD34 and CD31.

In addition, the Dexter type stromal cells had not previously beensubjected to Periodic Acid-Schiff (PAS) staining, which revealed astrong and uniform positivity by almost 100% of the MPCs studied. Thisindicates the presence of large stores of glycogen (FIG. 4D). Thepresence of glycogen (6) was confirmed by electron microscopy (see FIG.5). In this respect, MPCs are reminiscent of the glycogen-ladenreticular cells in the developing bone marrow of human fetuses (observedby L-T. Chen, L. Weiss, Blood 46, 389 (1975)). Glycogen deposition isviewed to be a developmentally regulated process during morphogenesis(H. Ohshima, J. Wartiovaara, I. Thesleff, Cell Tissue Res. 297, 271(1999)).

The MPCs also exhibited cytoplasm compartmentalization into endoplasmand ectoplasm. This morphologic finding sheds light on their internalarchitecture because of correlation of restricted localization ofglycogen and smooth muscle actin to ectoplasm; and the restrictedlocalization of acid phosphatase, alkaline phosphotase, Nile Red, OilRed O, fibronectin, and prolyl-4-hydrolase to endoplasm.

Additional sets of multiple mesenchymal lineage markers were assessed byNorthern blotting of unfractionated cells and purified MPCs to eliminateany observer bias that might be inherent in morphological assessment.FIGS. 6A-M represent different gene probes used for hybridization.

Compared to unfractionated cells, the purified nonhematopoietic cellsexpressed significantly higher levels of markers representing fat cells(adipsin, FIG. 6D); osteoblasts (osteoblast-specific cadherin-11,chondroitin sulfate, collagen type 1 and decorin, FIGS. 6E-H);fibroblasts (fibronectin, FIG. 6J); and smooth muscle cells (caldesmonand transgelin, FIGS. 6K-L).

Taken together, the morphologic, cytochemical, and immunocytochemicalresults (FIG. 4A-H and Table 1), and the Northern blotting data (FIGS.6A-M) indicate that the nonhematopoietic stromal cells of the Dextercultures co-express markers specific for at least four differentmesenchymal cell lineages. Using a variety of techniques, applicant hasdemonstrated that the MPCs co-express multilineage mesenchymal cellphenotypes, and in this respect the multi- or pluri-differentiated MPCsare distinct from the pluri-potential but undifferentiated, MSCs ofFriedenstein cultures (Prockop, Science 276, 71-74 (1997).

The nonhematopoietic cells of the present invention were purified fromthe macrophages, the dominant “contaminating” cell type, using a Percollgradient method developed by applicant. MPCs were purified by thefollowing process: cells from a Dexter-type culture were treated toobtain a cell suspension, the macrophages were removed, and the cellswere fractionated using discontinuous Percoll gradient centrifugation(FIG. 2). The isolated MPCs were then collected and washed.

The purity of the nonhematopoietic cells was demonstrated by a nearcomplete absence of two macrophage markers, CD68 and cathepsin B (asshown by Northern blotting data, FIGS. 6A and 6B). As a positivecontrol, bone marrow mononuclear cells rich in myelomonocytic cellsabundantly expressed CD68 (lanes 5 & 6, FIG. 6A). The Northern blotresults are consistent with a purity estimate of 95% (vs. 60% inunfractionated samples) based on morphology and immunocytochemicalstaining for CD68.

MPCs isolated to a purity of approximately 95% can be obtained usingmethods disclosed herein and Seshi B. et al., 2000, “Human Bone MarrowStromal Cell: Coexpession of Markers Specific for Multiple MesenchymalCell Lineages”, Blood Cells Mol Dis 26(3):234-246, which incorporatedherein by reference in its entirety. The remaining 5% of contaminatingcells (macrophages and hematopoietic cells) can be removed using methodsknown in the art, such as immunomagnetic separation (IMS) techniques,thereby achieving a purity of greater than 99%. Investigators havesuccessfully used immunomagnetic beads to separate and enrich carcinomacells from bone marrow and peripheral blood for some time (Naume et al.,1997, “Immunomagnetic Techniques for the Enrichment and Detection ofIsolated Breast Carcinoma Cells in Bone Marrow and Peripheral Blood”, J.Hematother., 6(2):103-114; Naume et al., 1998, “Increased Sensitivityfor Detection of Micrometastases in Bone-Marrow/Peripheral-BloodStem-Cell Products from Breast-Cancer Patients by NegativeImmunomagnetic Separation”, Int. J. Cancer, 78(5):556-560; Shibata, K.et al., 1998, “Detection of Ras Gene Mutations in Peripheral Blood ofCarcinoma Patients Using CD45 Immunomagnetic Separation and NestedMutant Allele Specification Amplification”, Int. J. Oncol.,12(6):1333-1338, each of which are incorporated by reference herein intheir entirety).

A purified source of MPCs is desirable for a number of reasons. Therelative ease with which large numbers of the MPCs can be purified andtheir distinctive phenotypic characteristics make them valuable targetsfor future investigations. Purified MPCs provide a sufficiently definedsystem to permit detailed elucidation of the role of bone marrow innormal and leukemic hematopoiesis in addition to aiding in bone marrowtransplantation.

Another major reason that purified cells are desirable is that Dextercultures also contain a significant percentage of highly immunogenicmacrophages that can cause onset of GvHD after transplantation. The MPCsof the present invention are purified to at least ˜95% free ofmacrophages and hematopoietic cells. Note the increased survival rate inSevere Combined Immunodeficiency Disease (SCID) mice that receivedpurified MPCs versus those that received unfractionated bone marrowstromal cells in FIGS. 13B-1 and 13B-2. This data establishes thatstromal cells in combination with engraftment or other similarprocedures enhances the effectiveness of the treatment.

The present invention also provides methods of enhancing the engraftmentof hematopoietic cells and of enhancing the engraftment of bone marrow.The hematopoietic support capacity of the Dexter-type cultures has beenrepeatedly demonstrated by a number of investigators. RT-PCR analysisshowed that Dexter cultures and Friedenstein cultures expressed asimilar pattern of cytokine and growth factor mRNAs; yet, Dextercultures were found to be more efficient than Friedenstein cultures inachieving preservation of hematopoietic progenitors (Majumdar, et al.,J. Cell. Physiol, 176, 57-66.). The pluri-differentiated MPC is capableof supporting hematopoiesis, as shown by its ability to expressrepresentative hematopoietic growth factors/cytokines, i.e., G-CSF andSCF as well as matrix receptors/hematopoietic cell adhesion molecules,i.e., ICAM-1, VCAM-1 and ALCAM (FIG. 7).

Clarification of the nature of the stromal cells and the ability topurify these cells makes it possible to use them as an adjuvant in bonemarrow transplantation following high-dose chemotherapy and radiationtherapy. These treatment modalities not only cause damage to thehematopoietic stem cells but also to the supportive stromal cells.However, because the bone marrow microenvironment is destroyed,hematopoietic progenitor cell engraftment is delayed until the stromalenvironment is restored. As a result, a critical aspect of the currentinvention is directed to the advantages of transplanting isolatedmesenchymal progenitor cells to accelerate the process of stromalreconstruction and ultimately bone marrow engraftment. The stromal cellspresent in the standard bone marrow transplant are not sufficient innumber and can be supplemented with the cultured MPCs of the presentinvention.

Yet another embodiment of the current invention provides the use of MPCtransplantation to major leukemic conditions, such as acute myeloidleukemia (AML), myelodysplastic syndromes (MDS), chronic myeloidleukemia (CML) and multiple myeloma (MM). This is based on applicant'sdetermination that bone marrow stromal cells in a leukemia patient arefunctionally and structurally defective, regardless of the damage causedby chemotherapy and radiation therapy. Such defects in bone marrowstromal cells are likely to aid and abet leukemia development.Alternatively, stromal cell defects could be secondarily induced bysurrounding leukemia cells, thus contributing to the loss ofhematopoietic support function of stromal cells and hematopoieticfailure, which is an invariable feature in leukemia. Regardless whetherthe observed stromal cell defects are primary or secondary to theleukemic process, by reason of their indisputable impact on normalhematopoiesis, these defects remain to be corrected to improve thehematopoietic function.

Stromal cells have never been carefully investigated in terms ofgenomics in view of the widespread belief that they represent aheterogeneous mixture of cell types. Tissue or cellular heterogeneitypresents a major challenge for the application of microarray technology.The purified stromal cells of the present invention represent a singlepluridifferentiated MPC which allows for genomic study of the stromalcells and the development of new, more objective diagnostic tools forpatients suffering from leukemia conditions.

The present invention provides a comprehensive phenotype of culturedbone marrow stromal cells at single cell level for the first time. Thesefindings pave the road for ultimate identification and investigation ofthese cells in fresh samples of marrow, normal as well as diseased, inwhich they occur at a low frequency and are extremely difficult to studyat the present time. The development of this phenotype forms the basisfor various diagnostic tests including a comprehensive test that can beused to screen for different abnormalities of bone marrow stromal cellsin various hematologic diseases and other diseases effecting stromalcells.

Results show that isolated single stromal cells simultaneously expresstranscripts for osteoblasts, fibroblasts, muscle cells and adipocytes.Furthermore, there is shown that isolated single stromal cellssimultaneously express transcripts for epithelial cells and neural/glialcells as well as transcripts for CD45, CD19, CD10, CD79a, andrepresentative proto-oncogenes and transcription factors, typicallyknown to be affiliated with normal and neoplastic hematopoietic cells.These findings are evidence of existence of a progenitor cell that iscommon to nonhematopoietic mesenchymal cells and hematopoietic cells,particularly B-lymphocytes. “Lineage burst” characterized bysimultaneous activation of diverse differentiation pathways within thesame cell appears to be the signature profile of a stromal cell,indicating that a “pluripotent” cell is “pluridifferentiated” at themolecular level. That is, prior to a selective and full-fledged lineagedifferentiation, progenitors express genes associated with multiplelineages to which they might possibly commit, thus providing insightinto the molecular basis of cellular plasticity.

Transcriptomic analysis has been undeniably contributing to themolecular definition of new disease categories with demonstrabletherapeutic benefit. The present invention contributes to the furtherdefinition of the stromal cell by refining its molecular signature. Thein vivo identification of the stromal cell and its possible ontogenicvariants as they might occur in different hematological diseases andsubsequent targeting of these cells holds the key to ultimately treatingsome, if not all, of these diseases.

By comprehensively defining the gene expression profile of these cells,the present invention demonstrates the technical applicability ofsingle-cell genomics toward understanding the physiology and pathologyof both hematopoietic and nonhematopoietic microenvironments.Classically, the adventitial reticular cells located on the abluminalside of the vascular endothelium within the bone marrow microenvironmentwere thought to represent the stromal cells or their precursors. As withhematopoietic stem or progenitor cells, the stromal progenitor cells arerare in bone marrow occurring at an estimated frequency of 1 in 10⁵nucleated cells. Cultured stromal cells represent the progeny of thestromal cell, and not necessarily the stromal cell itself, for which noin vivo assay exists as yet. The technology of single-cell genomics andthe blueprint as described in the present invention allows screening forthe abnormalities of bone marrow stromal cells in fresh marrow samplesthat reflect on the ultimate in vivo context.

The ability to purify culture-expanded MPCs from both normal individualsand patients afflicted with various leukemias also allows testing of thehematopoietic supportive role of MPCs in mice models. These systemsprovide an in vivo model in which to examine the role of human bonemarrow microenvironment in normal and leukemic hematopoiesis.

The SCID mouse model is an ideal system in which to investigate MPCfunction. Engraftment of human hematopoietic progenitors in SCID micehas required either coadministration of exogenous human cytokines, orcotransplantation of human bone marrow plugs or bone fragments. Asdisclosed herein MPCs are a convenient, new source for human bone marrowstromal cells for enhancing transplantation that does not requirecytokines, bone fragment, or marrow.

Unlike prior methods, the isolated MPCs of the present invention supporthuman hematopoiesis in the SCID mouse model as effectively as wholemarrow stroma. The transplantation of human marrow mononuclear cellscombined with purified MPCs results in dramatically vigorous engraftmentof human cells in spleen, bone marrow, liver, pancreas, lungs, stomach,and paravertebral neuronal ganglia of SCID mice (FIGS. 10A-H and FIGS.11A-C). By contrast, mice receiving human bone marrow mononuclear cellsalone or MPCs alone expectedly showed no detectable evidence of humanhematopoietic cell engraftment (FIGS. 13A-1, 13A-2, 13B-1, and 13B-2).

The present invention also provides for a method of preventing ortreating GvHD. The highest mortality rate, FIG. 13B, was observed inmice receiving the unpurified whole marrow stroma and the bone marrowmononuclear cells. The increased mortality observed is related to thepresence of highly immunogenic macrophages and consequent GvHD. The micewith the highest survival rate, shown in FIG. 13A, were the micereceiving purified MPCs and bone marrow mononuclear cells.

Notably, there is discrete TUNEL-positive nuclei in the liver of theexpired mouse in FIG. 14B and complete absence of staining in the liverof the surviving mouse (see FIG. 14A). While some ill-defined globulesof staining are observed in the spleen of the mouse that survived, thenuclear integrity of most of the cells is well preserved suggestingminimal or no apoptosis (FIG. 14C). In contrast, the dead mouse spleen(FIG. 14D) showed extensive TUNEL positivity precluding accurateinterpretation. Control mouse liver and spleen showed results similar tothose of the mouse that survived.

The above results indicate that purified MPCs can support humanhematopoiesis in SCID mice as effectively as whole marrow stroma.Equally important is that the purified MPCs increased the survival rate.The evidence shows that the increased survival is due to a reduction inGvHD.

Allogeneic bone marrow transplantation is the preferred method oftreatment for a variety of malignant and genetic diseases of the bloodand blood forming cells. However, failure of hematopoietic cellengraftment can occur for a number of reasons. These include,microenvironmental defects as part of the underlying disease itself(e.g., aplastic anemia), and/or stromal cell damage caused bychemoradiotherapy and/or microenvironmental damage as part of GvHD whichis a dreaded complication following bone marrow transplantation. InGvHD, donor T cells present in the hematopoietic cell graft destroy hosttissues. GvHD can involve multiple organs such as skin, liver, GI systemetc. The current treatment modalities for preventing or treating graftfailure or GvHD are cumbersome, costly and involve some form ofimmunosuppression. Stromal cell lesions, either primary to the diseaseprocess or secondarily induced by allogeneic bone marrowtransplantation, play a prominent role in the success or failure of thehematopoietic cell graft. Cotransplantation of MPC not only enhanceshematopoietic cell engraftment but also prolongs the life of graftrecipients by minimizing GvHD. Co-transplantation of healthy,culture-expanded MPC is a viable option in these situations. The humanbone marrow used in the Dexter-type cultures of the present inventioncan be obtained from a number of different sources in accordance withthe procedures known in the art, including from plugs of femoral headcancerous bone pieces or from aspirated marrow. The cells used in theDexter culture can be autologous, from the tissue donor, or from otherindividuals.

Modes of administration of MPCs include, but are not limited to,systemic intravenous injection and injection directly to the intendedsite of activity. The MPCs can be administered by any convenient route,for example by infusion or bolus injection, and can be administeredtogether with other biologically active agents. Administration ispreferably systemic.

The methods of the present invention can be altered, particularly by (1)increasing or decreasing the time interval between administering MPCsand implanting the tissue, cells, or implanting the organs; (2)increasing or decreasing the amount of MPCs administered; (3) varyingthe number of MPC administrations; (4) varying the method of delivery ofthe MPCs; and/or (5) varying the source of MPCs.

The MPC preparations are used in an amount effective to promoteengraftment of hematopoietic progenitor cells or bone marrow cells; orfor the treatment or prevention of GvHD in the recipient. Thepharmaceutically effective amount for the purposes herein is thusdetermined by such considerations as are known in the art. In general,such amounts are typically at least 1×10⁴ MPCs per kg of body weight andmost generally need not be more than 7×10⁵ MPCs/kg.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of MPCs and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to McCoy's medium, saline, buffered saline,dextrose, water, and combinations thereof. In one embodiment, thepharmaceutically acceptable carrier is pharmaceutical grade water orsaline. The formulation should suit the method of administration as isknown by those of skill in the art. The composition may be liquid (suchas an injectable cell suspension), semi-solid, or solid (such as atissue scaffold).

In one embodiment, the MPC preparation or composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition can alsoinclude a local anesthetic to ameliorate any pain at the site of theinjection. Generally, the ingredients are supplied either separately ormixed together in unit dosage form, for example, as a cryopreservedconcentrate in a hermetically sealed container such as an ampouleindicating the quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients can bemixed prior to administration.

The pharmaceutical compositions of the subject invention can beformulated according to known methods for preparing pharmaceuticallyuseful compositions. Formulations are described in a number of sourceswhich are well known and readily available to those skilled in the art.For example, Remington's Pharmaceutical Sciences (Martin E W, EastonPa., Mack Publishing Company, 19^(th) ed., 1995)) describes formulationswhich can be used in connection with the subject invention. Formulationssuitable for parenteral administration include, for example, aqueoussterile injection solutions, which may contain antioxidants, buffers,bacteriostats, and solutes which render the formulation isotonic withthe blood of the intended recipient; and aqueous and nonaqueous sterilesuspensions which may include suspending agents and thickening agents.The compositions may be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in a freezedried (lyophilized) condition requiring only the condition of thesterile liquid carrier, for example, water for injections, prior to use.It should be understood that in addition to the ingredients particularlymentioned above, the compositions of the subject invention can includeother agents conventional in the art having regard to the type offormulation in question. For example, in addition to isolatedmesenchymal progenitor cells of the subject invention (MPCs) and apharmaceutically acceptable carrier, the composition may furthercomprise cells other than isolated mesenchymal progenitor cells, ortissue, for co-transplantation. As used herein, the terms“transplanting”, “implanting”, “administering”, and grammaticalvariations thereof are used herein interchangeably to refer to thedelivery of the particular agent (e.g. cells or composition)systemically or to a target site within the subject.

The MPCs of the present invention may be administered to a subject, suchas a human or non-human mammal (e.g., the mouse model of the subjectinvention, in conjunction with other therapeutic agents, such asanti-cancer agents, cytotoxic agents, and/or chemotherapeutic agents.The MPCs of the present invention may be administered to the subject inconjunction with, or in the absence of, immunosuppressive treatment.

As used herein, the term “anti-cancer agent” refers to a substance ortreatment that inhibits the function of cancer cells, inhibits theirformation, and/or causes their destruction in vitro or in vivo. Examplesinclude, but are not limited to, cytotoxic agents (e.g., 5-fluorouracil,TAXOL) and anti-signaling agents (e.g., the PI3K inhibitor LY).

As used herein, the term “cytotoxic agent” refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells in vitro and/or in vivo. The term is intended to includeradioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², and radioactive isotopes of Lu), chemotherapeutic agents,toxins such as small molecule toxins or enzymatically active toxins ofbacterial, fungal, plant or animal origin, and antibodies, includingfragments and/or variants thereof.

As used herein, the term “chemotherapeutic agent” is a chemical compounduseful in the treatment of cancer, such as, for example, taxanes, e.g.,paclitaxel (TAXOL, BRISTOL-MYERS SQUIBB Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE, Rhone-Poulenc Rorer, Antony, France), chlorambucil,vincristine, vinblastine, anti-estrogens including for exampletamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (Fareston), and anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin, etc.

The terms “comprising”, “consisting of”, and “consisting essentially of”are defined according to their standard meaning. The terms may besubstituted for one another throughout the instant application in orderto attach the specific meaning associated with each term. The phrases“isolated” or “biologically pure” refer to material that issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state. Thus,isolated pluri-differentiated mesenchymal progenitor cells of thepresent invention (MPCs) preferably do not contain materials normallyassociated with the cells in their in situ environment, such ashematopoietic cells and macrophages. In one embodiment, the MPCs are atleast 95% pure. In another embodiment, the MPCs are at least 99% pure.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the development or spread ofcancer. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, alleviation of symptoms,diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. Those subjects (e.g., human or veterinary patients) in needof treatment include those already with the condition or disorder aswell as those prone to have the condition or disorder or those in whichthe condition or disorder is to be prevented.

As used herein, the term “(therapeutically) effective amount” refers toan amount of an agent (e.g., a cell or composition) effective to treat adisease or disorder in a human or non-human mammal.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural reference unless the contextclearly dictates otherwise. Thus, for example, a reference to “anisolated pluri-differentiated mesenchymal progenitor cells” or “an MPC”includes more than one such cell.

The present invention paves the way for applications of mesenchymalprogenitor cells in the field of transplantation with respect tohematopoietic support, immunoregulation, and graft facilitation. MPCscan be used as a supporting cell type in bone marrow transplantation,particularly in diseases where defects in the hematopoietic stromalmicroenvironment are believed to prevail, such as aplastic anemia,myelofibrosis, and bone marrow failure following high dose chemotherapyand radiation therapy.

Another aspect of the invention provides a method for diagnosing variousdisease states in mammals by identifying new diagnostic markers,specifically the classification and diagnosis of leukemia. Prior to thepresent invention, stromal cells were not carefully investigated interms of genomics because of the widespread belief that they represent aheterogeneous mixture of cell types and cellular heterogeneity presentssignificant challenges for the application of genetic analysis such asmicroarray technology. The isolated MPCs of the present inventionrepresent a single cell type and allow for genomic study of the stromalcells.

Using the methods of the present invention, it has been determined thatbone marrow stromal cells in leukemia patients are functionally andstructurally defective regardless of the damage caused by chemotherapyand radiation therapy. Given the almost 25 year history and intenseinterest in bone marrow stromal cell cultures, previous documentation ofstromal cell abnormalities has been disappointingly low (Martinez &Martinez, Exp. Hematol 11:522-26 (1983); Budak-Alpdogan, et al., Am. J.Hematol, 62:212-20 (1999); Nagao, et al., Blood, 61:589-92 (1983);Peled, et al., Exp. Hematol 24:728-37 (1996); Bhatia, et al., Blood85:3636-45 (1995); Agarwal, et al., Blood 85:1306-12 (1995); Diana, etal., Blood 96:357a (2000)). By identifying gene sets that are unique toa given state, these differences in the stromal cells can be utilizedfor diagnostic purposes.

In one embodiment of the invention, isolated MPCs from a patient areassayed for expression of a large number of genes. The gene expressionprofile is projected into a profile of gene set expression valuesaccording to the definition of gene sets. A reference databasecontaining a number of reference projected profiles is also created fromthe isolated MPCs of patients with known states, such as normal andvarious leukemic disease states. The projected profile is then comparedwith the reference database containing the reference projected profiles.If the projected profile of the patient matches best with the profile ofa particular disease state in the database, the patient is diagnosed ashaving such disease state. Various computer systems and software (seeExample 5) can be utilized for implementing the analytical methods ofthis invention and are apparent to one of skill in the art. Some ofthese software programs include Cluster & TreeView (Stanford),GeneCluster (MIT/Whitehead institute), Array Explorer (SpotFire Inc.)and GENESPRING (Silicon Genetics Inc.) (for computer systems andsoftware, see also U.S. Pat. No. 6,203,987, which is incorporated hereinby reference in its entirety).

The methods of the present invention can also be useful for monitoringthe progression of diseases and the effectiveness of treatments. Forexample, by comparing the projected profile prior to treatment with theprofile after treatment.

One aspect of the present invention provides methods for therapeutic anddrug discovery utilizing bone marrow derived isolated mesenchymalprogenitor cells. The present invention can be utilized to identifystromal cell genes that can be therapeutic targets for improvement ofnormal hematopoietic function, which is constantly compromised, inleukemic patients. In one embodiment, gene sets are defined usingcluster analysis. The genes within a gene set are indicated aspotentially co-regulated under the conditions of interest. Co-regulatedgenes are further explored as potentially being involved in a regulatorypathway. Identification of genes involved in a regulatory pathwayprovides useful information for designing and screening new drugs.

Some embodiments of the present invention employ gene set definition andprojection to identify drug action pathways. In one embodiment, theexpression changes of a large number of genes in response to theapplication of a drug are measured. The expression change profile isprojected into a gene set expression change profile. In some cases, eachof the gene sets represents one particular pathway with a definedbiological purpose. By examining the change of gene sets, the actionpathway can be deciphered. In some other cases, the expression changeprofile is compared with a database of projected profiles obtained byperturbing many different pathways. If the projected profile is similarto a projected profile derived from a known perturbation, the actionpathway of the drug is indicated as similar to the known perturbation.Identification of drug action pathways is useful for drug discovery.See, Stoughton and Friend, Methods for Identifying pathways of DrugAction, U.S. patent application Ser. No. 09/074,983; U.S. PatentPublication 2001/0018182, filed Feb. 14, 2001; U.S. Patent Publication2002/0128781, filed Jan. 28, 2002; U.S. Patent Publication 2003/0093227,filed Nov. 4, 2002; U.S. Pat. No. 6,468,476, filed Oct. 27, 1999; U.S.Pat. No. 6,351,712, filed Dec. 28, 1998; and U.S. Pat. No. 6,218,122,filed Jun. 16, 1999, which are incorporated herein by reference in theirentirety.

The present invention provides a genomics strategy method foridentifying genes differentially expressed in MPCs. The method beginswith the preparation of total RNA from MPC samples, which leads to thegeneration of cDNA. From the cDNA, ds DNA can be prepared for in vitrotranscription into cRNA. The cRNA is then fragmented for thehybridization of target RNA to a microarray of known genes (Affymetrixgenechip containing DNA from ˜12,000 known human genes, e.g., U95Aoligonucleotide microarray). Finally, analysis of differentiallyexpressed genes is accomplished using appropriate software (GENESPRING)to discern the patterns of gene expression or genomic signatures by agiven MPC type (e.g., up-regulation or down-regulation).

Up-regulated and down-regulated gene sets for a given disease-associatedor cytokine-stimulated MPC are combined. The combination enables thoseof skill in the art to identify gene sets with minimal number ofelements that are unique to a given MPC type with a capability todiscriminate one MPC type from another (this can be accomplished bymeans of a series of Venn diagrams and lists of required genes obtainedvia GENESPRING). Such gene sets are of immense diagnostic value as theycan be routinely used in assays that are simpler than microarrayanalysis (for example “real-time” quantitative PCR). Such gene sets alsoprovide insights into pathogenesis and targets for design of new drugs.For example, the method allows one to establish transcriptional profilesof MPC genes that are pathologically altered.

Those of skill in the art can use the data and methods contained hereinfor the following: a) study select gene or sets of genes that arerelevant to hematopoietic disease conditions by using relativelyinexpensive but low-throughput technologies such as Northern blotting,RNase protection assays and/or PCR intended for gene expressionanalysis; b) identify newer drug targets and diagnostic markers relevantto specific diseases, such as MM or CML etc depending on the researchinterests of the individual investigators.

The present invention also provides a large-format 2-D gelelectrophoretic system for the reproducible separation of MPC proteinsand for preparing 2-D PAGE protein maps for normal bone marrow-derivedMPCs (untreated and treated with representative cytokines, e.g., TNF-αand/or IL-4) and for MPCs derived from patients with representativepre-leukemic/premalignant and leukemic/malignant conditions. Thepre-leukemic conditions include myelodysplastic syndromes (MDS) and theleukemic conditions include chronic myeloid leukemia (CML), acutemyeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acutelymphocytic leukemia (ALL), and multiple myeloma (MM). The proteinsamples consist of culture supernatants/secreted proteins, extracellularmatrix (ECM) proteins, plasma membrane proteins solubilized using athree-step differential extraction protocol, employing conditions ofprogressively increasing solubility; and whole cell lysate proteinssimilarly solubilized using the three-step differential extractionprotocol. This subproteome approach not only simplifies the 2-D PAGEelectrophoretic protein patterns but also reveals additional proteins,which would otherwise have gone undetected.

The 2-D system described herein utilizes an immobilized pH gradient gel(pH 4-7) in the first dimension and a mini non-denaturing buthigh-resolution lithium dodecyl sulfate-polyacrylamide gelelectrophoresis (LDS-PAGE) in the second dimension. As identified bysilver staining, this system has resolved greater than 800 protein spotsin a pH interval of 2.5 units (4.25-6.75, the isoelectric pH range formost of plasma membrane proteins to migrate) and a molecular mass rangeof 10-150 kDa. Equally important, the system is compatible with highsample loads (up to 1.5 to 2.0 mg of total protein in up to 350 μlsample volume). All the protein species identifiable by a silver stainthat is compatible with subsequent mass spectrometric analysis have beenanalyzed by a 2-D gel software with respect to isoelectric point,molecular weight and mass abundance. The lectin-binding status of theseproteins has also been determined by lectin blotting. Lectin blots andWestern blots have subsequently been stained by a gold stain fordetection of total proteins on the same PVDF membrane. Althoughgold-staining of the Western blot is not as sensitive as silver-stainingof the gel, gold-staining of the Western blot generates the necessarylandmarks for alignment with the silver stained gel, facilitatingexcision of spots of interest from the gel for identification byMALDI-MS. Representative protein spots were excised from gel andsubjected to mass spectrometric profiling (MALDI-MS) and/or sequencing(Nano ESI MS/MS) with subsequent database searching, resulting in aproductive identification of ten proteins.

As used herein, the term “expression products” refers to ribonucleicacid (RNA) or polypeptide products transcribed or translated,respectively, from a genome or other genetic element. Commonly,expression products are associated with genes having biologicalproperties. Thus, the term “gene” refers to a nucleic acid sequenceassociated with a biological properties, e.g., encoding a gene productwith physiologic properties. A gene optionally includes sequenceinformation required for expression of the gene (e.g., promoters,enhancers, etc.).

As used herein, the terms “expression” or “gene expression” refer totranscription of a gene into an RNA product, and optionally totranslation into one or more polypeptide sequences. The term“transcription” refers to the process of copying a DNA sequence of agene into an RNA product, generally conducted by a DNA-directed RNApolymerase using DNA as a template.

As used herein, the term “nucleic acid” refers to a polymer ofribonucleic acids or deoxyribonucleic acids, including RNA, mRNA, rRNA,tRNA, small nuclear RNAs, cDNA, DNA, PNA, RNA/DNA copolymers, oranalogues thereof. Nucleic acid may be obtained from a cellular extract,genomic or extragenomic DNA, viral RNA or DNA, orartificially/chemically synthesized molecules.

As used herein, the term “RNA” refers to a polymer of ribonucleic acids,including RNA, mRNA, rRNA, tRNA, and small nuclear RNAs, as well as toRNAs that comprise ribonucleotide analogues to natural ribonucleic acidresidues, such as 2-O-methylated residues.

As used herein, the term “cDNA” refers to complementary or “copy” DNA.Generally cDNA is synthesized by a DNA polymerase using any type of RNAmolecule (e.g., typically mRNA) as a template. Alternatively, the cDNAcan be obtained by directed chemical syntheses.

As used herein, the term “amplified product” or “amplified nucleic acid”refers to a nucleic acid generated by any method of nucleic acidamplification.

As used herein, the term “complementary” refers to nucleic acidsequences capable of base-pairing according to the standard Watson-Crickcomplementary rules, or being capable of hybridizing to a particularnucleic acid segment under relatively stringent conditions. Nucleic acidpolymers are optionally complementary across only portions of theirentire sequences.

As used herein, the term “hybridization” refers to duplex formationbetween two or more polynucleotides, e.g., to form a double-strandednucleic acid. The ability of two regions of complementarity to hybridizeand remain together depends of the length and continuity of thecomplementary regions, and the stringency of hybridization conditions.

As used herein, the term “defined sequence probe” is a nucleic acidprobe having a single polynucleotide sequence.

As used herein, the term “synthetic probe” is used to indicate that theprobe is produced by one or more synthetic or artificial manipulations,e.g., restriction digestion, amplification, oligonucleotide synthesis,cDNA synthesis, and the like.

As used herein, the term “label” refers to any detectable moiety. Alabel may be used to distinguish a particular nucleic acid from othersthat are unlabeled, or labeled differently, or the label may be used toenhance detection.

As used herein, the term “primer” refers to any nucleic acid that iscapable of hybridizing at its 3′ end to a complementary nucleic acidmolecule, and that provides a free 3′ hydroxyl terminus which can beextended by a nucleic acid polymerase.

As used herein, the term “template” refers to any nucleic acid polymerthat can serve as a sequence that can be copied into a complementarysequence by the action of, for example, a polymerase enzyme.

As used herein, the term “target,” “target sequence,” or “target genesequence” refers to a specific nucleic acid sequence, the presence,absence or abundance of which is to be determined. In a preferredembodiment of the invention, it is a unique sequence within the mRNA ofan expressed gene.

As used herein, the term “gene expression data” refers to one or moresets of data that contain information regarding different aspects ofgene expression. The data set optionally includes information regarding:the presence of target-transcripts in cell or cell-derived samples; therelative and absolute abundance levels of target transcripts; theability of various treatments to induce expression of specific genes;and the ability of various treatments to change expression of specificgenes to different levels.

As used herein, the term “quantitating” means to assign a numericalvalue, e.g., to a hybridization signal. Typically, quantitating involvesmeasuring the intensity of a signal and assigning a corresponding valueon a linear or exponential numerical scale.

As used herein, the term “algorithm” refers to a set of rules fordescribing a biological condition. The rule set may be definedexclusively algebraically but may also include alternative or multipledecision points requiring domain-specific knowledge, expertinterpretation or other clinical indicators.

As used herein, the term “baseline profile data set” refers to a set ofvalues associated with constituents of a gene expression panel resultingfrom evaluation of a biological sample (or population of samples) undera desired biological condition that is used for mathematically normativepurposes. The desired biological condition may be, for example, thecondition of a subject (or population of subjects) before exposure to anagent or in the presence of an untreated disease or in the absence of adisease. Alternatively, or in addition, the desired biological conditionmay be health of a subject or a population of subjects. Alternatively,or in addition, the desired biological condition may be that associatedwith a population subjects selected on the basis of at least one of agegroup, gender, ethnicity, geographic location, diet, medical disorder,clinical indicator, medication, physical activity, body mass, andenvironmental exposure.

As used herein, the term “biological condition” of a subject is thecondition of the subject in a pertinent realm that is under observation,and such realm may include any aspect of the subject capable of beingmonitored for change in condition, such as health, disease includingcancer; trauma; aging; infection; tissue degeneration; developmentalsteps; physical fitness; obesity, and mood. As can be seen, a conditionin this context may be chronic or acute or simply transient. Moreover, atargeted biological condition may be manifest throughout the organism orpopulation of cells or may be restricted to a specific organ (such asskin, heart, eye or blood), but in either case, the condition may bemonitored directly by a sample of the affected population of cells orindirectly by a sample derived elsewhere from the subject. The term“biological condition” includes a “physiological condition, such as ahematologic disease or other disease affecting stromal cells, andleukemic states such as pre-leukemic conditions (e.g., myelodysplasticsyndrome (MDS), overt leukemia, lymphoma, acute myeloid leukemia (AML),chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL),chronic lymphocyte leukemia (CLL), and multiple myeloma (MM).

As used herein, the term “calibrated profile data set” is a function ofa member of a first profile data set and a corresponding member of abaseline profile data set for a given constituent in a panel.

As used herein, a “clinical indicator” is any physiological datum usedalone or in conjunction with other data in evaluating the physiologicalcondition of a collection of cells or of an organism. This term includespre-clinical indicators.

As used herein, to “derive” or “prepare” a profile data set, such as agene expression profile, from a sample includes determining a set ofvalues associated with constituents of a gene expression panel either(i) by direct measurement of such constituents in a biological sample or(ii) by measurement of such constituents in a second biological samplethat has been exposed to the original sample or to matter derived fromthe original sample.

As used herein, the term “distinct RNA or protein constituent” in apanel of constituents is a distinct expressed product of a gene, whetherRNA or protein. An “expression” product of a gene includes the geneproduct whether RNA or protein resulting from translation of themessenger RNA.

As used herein, the term “gene expression panel” refers to anexperimentally verified set of constituents, each constituent being adistinct expressed product of a gene, whether RNA or protein, whereinconstituents of the set are selected so that their measurement providesa measurement of a targeted biological condition.

As used herein, the term “gene expression profile” refers to a set ofvalues associated with constituents of a gene set or gene expressionpanel resulting from evaluation of a biological sample (or population ofsamples). For example, a gene expression profile can have a minimumnumber of values selected from the group consisting of 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, or more. Accordingto the methods of the invention, two or more gene expression profilescan be compared. The most similar reference profile can be selected byweighting a comparison value for each value of the plurality using aweight value associated with the corresponding gene.

As used herein, the term “index” is an arithmetically or mathematicallyderived numerical characteristic developed for aid in simplifying ordisclosing or informing the analysis of more complex quantitativeinformation. A disease or population index may be determined by theapplication of a specific algorithm to a plurality of subjects orsamples with a common biological condition.

A “large number” of data sets based on a common panel of genes is anumber of data sets sufficiently large to permit a statisticallysignificant conclusion to be drawn with respect to an instance of a dataset based on the same panel.

As used herein, a “normative” condition of a subject to whom acomposition is to be administered means the condition of a subjectbefore administration, even if the subject happens to be suffering froma disease.

As used herein, a “panel” of genes means a set of genes (a “gene set”)including at least two constituents.

A “sample” from a subject may include a single pluri-differentiatedmesenchymal progenitor cell or a plurality of pluridifferentiatedmesenchymal progenitor cells taken from the subject, by any means knownin the art. The sample may be obtained directly from the subject or fromprimary culture, such as Dexter culture.

As used herein, the term “signature profile” means an experimentallyverified subset of a gene expression profile selected to discriminate abiological condition, agent or physiological mechanism of action.

As used herein, the term “signature panel” refers to a subset of a geneexpression panel, the constituents of which are selected to permitdiscrimination of a biological condition, agent or physiologicalmechanism of action.

As used herein, the term “therapy” includes all interventions whetherbiological, chemical, physical, metaphysical, or combination of theforegoing, intended to sustain or alter the monitored biologicalcondition of a subject.

Gene expression panels may be used for measurement of therapeuticefficacy of natural or synthetic compositions or stimuli that may beformulated individually or in combinations or mixtures for a range oftargeted physiological conditions; prediction of toxicological effectsand dose effectiveness of a composition or mixture of compositions foran individual or in a population; determination of how two or moredifferent agents administered in a single treatment might interact so asto detect any of synergistic, additive, negative, neutral or toxicactivity; performing pre-clinical and clinical trials by providing newcriteria for pre-selecting subjects according to informative profiledata sets for revealing disease status; and conducting preliminarydosage studies for these patients prior to conducting phase 1 or 2trials. These gene expression panels may be employed with respect tosamples derived from subjects in order to evaluate their biologicalcondition.

A gene expression panel is preferably selected in a manner so thatquantitative measurement of RNA or protein constituents in the panelconstitutes a measurement of a biological condition (such as a leukemicstate) of a subject. In one kind of arrangement, a calibrated profiledata set is employed. Each member of the calibrated profile data set isa function of (i) a measure of a distinct constituent of a geneexpression panel and (ii) a baseline quantity. Further informationregarding derivation, analysis, and comparison of gene expressionprofiles and gene expression panels are disclosed in U.S. PatentPublication 2004/0133352 (Bevilacqua et al.), filed Nov. 8, 2002, andU.S. Patent Publication 2004/0132050 (Monforte et al.), filed Jul. 16,2003, which are incorporated herein by reference in their entirety.

The practice of the present invention can employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA technology, electrophysiology, and pharmacology, thatare within the skill of the art. Such techniques are explained fully inthe literature (see, e.g., Sambrook, Fritsch & Maniatis, MolecularCloning: A Laboratory Manual, Second Edition (1989); DNA Cloning, Vols.I and II (D. N. Glover ed. 1985); Perbal, B., A Practical Guide toMolecular Cloning (1984); the series, Methods In Enzymology (S. Colowickand N. Kaplan eds., Academic Press, Inc.); Transcription and Translation(Hames et al. eds. 1984); Gene Transfer Vectors For Mammalian Cells (J.H. Miller et al. eds. (1987) Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.); Scopes, Protein Purification Principles and Practice (2nded., Springer-Verlag); and PCR: A Practical Approach (McPherson et al.eds. (1991) IRL Press)).

Each of the following applications are incorporated herein by referencein their entirety, including all nucleic acid sequences, amino acidsequences, figures, tables, and claims: U.S. Provisional PatentApplication No. 60/486,077, filed Jul. 9, 2003; U.S. patent applicationSer. No. 10/263,419, filed Oct. 3, 2002; U.S. Provisional PatentApplication No. 60/327,140, filed Oct. 3, 2001; U.S. Provisional PatentApplication No. 60/334,277, filed Nov. 28, 2001; U.S. Provisional PatentApplication No. 60/352,636, filed Jan. 28, 2002; U.S. Provisional PatentApplication No. 60/412,450, filed Sep. 20, 2002; U.S. patent applicationSer. No. 09/914,508, filed Nov. 7, 2001 (which is a National StageApplication of International Application Number PCT/US01/16408, filedMay 21, 2001); U.S. Provisional Patent Application No. 60/277,700, filedMar. 21, 2001; and U.S. Provisional Patent Application No. 60/209,245,filed Jun. 5, 2000.

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided for thepurpose of illustration only, and are not intended to be limiting unlessotherwise specified. Thus, the invention should in no way be construedas being limited to the following examples, but rather, should beconstrued to encompass any and all variations which become evident as aresult of the teaching provided herein.

EXAMPLES

The examples presented herein can be summarized as follows. The datadisclosed herein demonstrate that Dexter cultures consist of only threecell types macrophages (˜35%), hematopoietic cells (˜5%), andnonhematopoietic cells (˜60%). Using a percoll gradient centrifugationtechnique, the nonhematopoietic mesenchymal progenitor cells wereisolated, free of macrophages and hematopoietic cells. A variety oftechniques were used to identify the isolated cells as amulti-differentiated mesenchymal cell lineage co-expressing genesspecific for multiple mesenchymal cell lineages including adipocytes,osteoblasts, fibroblasts and muscle cells.

Evidence that this multi- or pluri-differentiated mesenchymal progenitorcell is capable of supporting hematopoiesis is shown by the expressionof a number of hematopoietic growth factors and extracellular matrixreceptors. The SCID mouse experimental data provides evidence that sincethe MPCs can be purified to near homogeneity (95%) with relative ease,MPCs can be of value for enhancing engraftment of hematopoietic stemcells and bone marrow transplants. Additionally, increased survival ratein the SCID mouse model indicates that isolated MPCs can also be usefulfor the treatment of GvHD. An example of the administration of bonemarrow cells and MPCs to breast cancer patients treated withchemotherapy is also provided.

A stepwise genomics strategy and an example of the genomic changesobserved in leukemia associated MPCs is also provided. Cluster analysiswas performed to show gene expression patterns in isolated MPCs of anormal individual and individuals with different leukemic conditions.The approach presented provides the basis for a new more objective meansto diagnose patients suffering from leukemic conditions.

Example 1 Isolation and Characterization of MPCs from Dexter-Type BoneMarrow Stromal Cell Culture Systems

Bone Marrow Culture: Bone marrow samples were obtained from posteriorsuperior iliac crest under general anesthesia for standard marrowtransplantation. Marrow stromal cell cultures were set up using theresidual cells recovered from the filters of Fenwal Bone MarrowCollection System after complete filtration, of the marrow samples. Thefilters were rinsed with phosphate-buffered saline without Ca²⁺ and Mg²⁺(PBS-CMF). The cell suspension was subjected to Ficoll gradientisolation of the mononuclear cells (bone marrow MNCs). The bone marrowMNCs were washed (×2) in PBS-CMF and suspended in McCoy's 5A with HEPESmedium containing 12.5% fetal bovine serum (FBS), 12.5% horse serum, 1μM/L hydrocortisone and 1% penicillin/streptomycin (for this studyMcCoy's complete medium) and cultured under standard stromal-cellculture conditions (FIG. 1) (Seshi, et al. Blood 83, 2399 (1994) andGartner, et al. Proc Natl Acad Sci USA 77, 4756 (1980). After two weeks,confluent stromal cell cultures were trypsinized (first passage),followed by splitting each T75 flask into two T150 flasks.

Morphologic and Phenotypic Characteristics of MPCs as Uncovered byStaining for Representative Mesenchymal Cell Lineage Markers:

Two weeks after the first passage (above), confluent stromal cells wereagain trypsinized. Cytospins were prepared using aliquots ofunfractionated cells for performance of various cytological,cytochemical and immunocytochemical stains.

Reactivity patterns of the bone marrow culture cells are outlined inTable 1. FIGS. 4A-E illustrate morphologic and phenotypiccharacteristics, as uncovered by staining for representative celllineage markers. As illustrated in Table 1 and FIGS. 3 and 4A,Wright-Giemsa staining revealed three morphologically identifiable cellpopulations in Dexter type stromal cell cultures, macrophages,hematopoietic cells, and nonhematopoietic cells (labeled 4, 3, and 5,respectively).

The identity of macrophages was confirmed by immunostain using anti-CD68antibody (FIG. 4B) and cytochemical stains for acid phosphatase andSudan black. The identity of hematopoietic cells (including macrophages)was confirmed by immunostain using anti-CD45 antibody (FIG. 4C).

The remaining nonhematopoietic cells stained intensely positive forPeriodic acid-Schiff, which was diastase sensitive, signifying thepresence of large stores of glycogen (FIG. 4D). The presence of glycogen(6) was confirmed by electron microscopy (see FIG. 5). In this respect,MPCs are reminiscent of the glycogen-laden reticular cells in thedeveloping bone marrow of human fetuses (observed by L-T. Chen, L.Weiss, Blood 46, 389 (1975)). Glycogen deposition is viewed to be adevelopmentally regulated process during morphogenesis (H. Ohshima, J.Wartiovaara, I. Thesleff, Cell Tissue Res. 297, 271 (1999)).

In terms of lineage markers, up to 100% of the nonhematopoietic cellsexpressed two fat cell markers (Nile Red (FIG. 4E) and Oil Red O); anosteoblast marker (alkaline phosphatase (FIG. 4F)); and two fibroblastmarkers (fibronectin (FIG. 4G) and prolyl-4-hydroxylase). Greater than85% of the nonhematopoietic cells were also positive for a musclemarker, actin (FIG. 4H). There was no evidence of expression ofendothelial cell differentiation, as judged by immunohistochemicalstaining for CD34 and CD31 (data not shown).

The results indicate that the nonhematopoietic cells of the Dextercultures are in fact a single, pluri-differentiated cell typeco-expressing multiple mesenchymal cell lineage markers. Thepluri-differentiated mesenchymal progenitor cells reported here are tobe distinguished from the pluri-potential, but undifferentiated, MSCsthat are generated in the absence of hematopoietic cells, such as inFriedenstein-type cultures.

TABLE 1 Reactivity patterns of bone marrow stromal cells based oncytological, cytochemical and immunocytochemical stains*,*** TestHematopoietic Mesenchymal Figure Utilized Macrophages Cells ProgenitorCells 1 3 and 4A Wright- Large cells Small cells Large cells with aGiemsa with a small with minimal relatively irregular (Harleco) roundnucleus amount of nucleus & & foamy cytoplasm: cytoplasm cytoplasm: 5%of total compartmentalized 35% of total cells into ectoplasm and cellsendoplasm: 60% of total cells 2 4D Periodic acid- 0 0 ~100% MPCs: Schiff(PAS) staining restricted (Sigma) to ectoplasm in a ring-like fashion;and completely abolished by diastase digestion 3 4C CD45 (Dako, 100%100% HCs 0 PD7/26 & macrophages 2B11) (MΦ) 4 4B CD68 100% MΦ 0 0(Immunotech, clone PG-M1) 5 Sudan Black ~100% MΦ 0 0 (Sigma) 6 Acid 100%MΦ; 0 100% MPCs: phosphatase positive positive granules (Sigma Kitgranules in moderate No. 387) packed amounts; staining throughoutrestricted to cytoplasm endoplasm 7 4E Nile Red 0 0 ~100% MPCs: (Sigma)staining restricted to endoplasm 8 Oil Red O 0 0 ~100% MPCs: (Sigma)staining restricted to endoplasm 9 4F Alkaline 0 0 ~100% MPCs:phosphatase variable number of (Sigma Kit positive granules; No. 85)staining restricted to endoplasm & plasma membrane** 10 4G Fibronectin 00 ~100% MPCs: (Immunotech, staining restricted clone 120.5) to endoplasm11 Prolyl-4- 0 0 ~100% MPCs: hydroxylase staining (Dako, clonepreferentially in 5B5) the endoplasm 12 4H Muscle actin 0 0 >85% MPCs:(Ventana, variable staining clone HUC 1- restricted to 1) ectoplasm *Thelineages of the markers tested above are: 3, hematopoietic cell marker;4, 5 and 6, monocyte/macrophage markers; 7 and 8, adipocyte markers; 9,osteoblast marker; 10 and 11, fibroblast markers; 12 muscle marker.**One earlier study (Simmons, et al., Nature 328, 429-432) interpretedthe localization of alkaline phosphatase staining as confined to theplasma membrane when in fact it is predominately present within theendoplasm (compare FIG. 1C of this reference with FIG. 4F). ***Whilewell-accepted mesenchymal lineage markers were used, these markers donot necessarily lend themselves to simultaneous assessment of the samecell. For example, muscle-specific actin antibody worked only onformalin-fixed, paraffin embedded material, whereas stains like alkalinephosphatase, Oil Red and Nile Red are not antibody based and involvevarying fixing and staining conditions. Thus, the evidence shows thatclose to 100% of members of a morphologically distinct population #express multiple lineage markers of interest.

Bone Marrow Mesenchymal Progenitor Cell (MPC) Purification: To furtherinvestigate the characteristics of the MPCs, the nonhematopoieticstromal cells were then purified from the macrophages (˜95% pure), thedominant “contaminating” cell type using the following method. Confluentmonolayers of stromal cells resulting from first passage, above, werewashed for three minutes in Ca²⁺/Mg²⁺ free Hanks' balanced saltsolution. Cells were incubated at room temperature for 45 minutes withintermittent mixing in serum-free McCoy's medium containing 10 mML-leucine methyl ester (LME, Sigma). LME is a lysosomotropic agent thatselectively kills and detaches macrophages. The detached macrophageswere removed by washing the monolayers twice in McCoy's complete medium,followed by trypsinization of the monolayers. The resulting single cellsuspensions were fractionated by discontinuous Percoll gradient (70%,50%, 30%, 20%, 10%) centrifugation at 800×G for 15 minutes at 4° C. in afixed angle rotor (Avanti-J25 Beckman centrifuge) (FIG. 2). Low-densitycells representing the macrophages resistant to detachment by LMEseparate as a band at the interface of serum and 10% Percoll and werediscarded (1). High-density nonhematopoietic cells representing MPCsform a layer in the region of 30-50% Percoll (2). These were collectedand washed twice by centrifugation through PBS-CMF. This protocol isconservatively expected to yield, >2.5×10⁶ MPCs per T-150 flask (i.e.,>50×10⁶ MPCs per batch of 20 flasks). The purity of these preparations,typically about 95%, was routinely monitored by Wright-Giemsa staining.

Northern Blotting: Additional sets of multiple mesenchymal lineagemarkers were assessed by Northern blotting to eliminate any observerbias that might be inherent in morphological assessment. FIGS. 6A-Mrepresent different gene probes used for hybridization. The sources ofthe gene probes employed and the major transcripts observed are outlinedin the brief description of the figures.

Total RNA was prepared by dissolving the high-density cell pellets inTrizol (Life-Technologies). Total RNA samples from unfractionatedstromal cells and BM MNCs were similarly prepared. The RNA samples wereelectrophoresed in a standard 1% agarose gel containing 2% formaldehydein MOPS/EDTA buffer and blotted onto Immobilon-Ny+ membrane. Probes werelabeled using Prime-A-Gene Kit (Promega) and a ³²P dCTP (NEN).Hybridization was performed at 65° C. in modified Church's hybridizationsolution using 3×10⁶ counts/ml in 10 ml (Millipore, 1998).

In FIGS. 6A-M, Northern blot analysis was performed side-by-side onfractionated stromal cells, non-hematopoietic cells freed ofmacrophages, and initial bone marrow mononuclear cell samples. Lanes 1and 2 represent total RNA samples (10 μg each) from unfractionatedstromal cells (subjects S1 and S2, respectively). Lanes 3 and 4represent total RNA samples (10 μg each) from purified stromal MPCs(subjects S1 and S2, respectively). Lanes 5 and 6 represent total RNAsamples (10 μg each) from bone marrow mononuclear cells, the startingcells for bone marrow cell cultures (subjects S3 and S4, respectively).

The large transcripts, especially of collagen (lane 1, FIG. 6G) andfibronectin (lane 1, FIG. 6J), in RNA extracted from unfractionatedstromal cells of subject 1 showed difficulty migrating into the gel.This observation correlates with the presence of an artifact ofunresolved positive material in lane 1, FIG. 6A. Since the RNA extractedfrom unfractionated stromal cells of the subject 2 did not present thisproblem (lane 2, FIG. 6G, FIG. 6J and FIG. 6A), the observation does notimpact on the overall interpretation of the results (see text). Thelineages of markers tested were: monocyte/macrophage markers, CD68 andcathepsin B; adipocyte marker, adipsin; osteoblast markers,osteoblast-specific cadherin-11, chondroitin sulfate proteoglycan 2,collagen type I alpha 1 and decorin; fibroblast marker, fibronectin;muscle markers, caldesmon and transgelin. Marker signals were normalizedto the amount of RNA loaded, which was based on densitometry of theGAPDH signals on the corresponding blot (Bio-Rad Model GS-700 ImagingDensitometer). Attenuation or enhancement of the marker signals in thepurified stromal MPCs (i.e., lanes 3 and 4) relative to unfractionatedstromal cells (i.e., lanes 1 and 2, respectively) is shown as fold A(decrease/increase) underneath the lanes 3 and 4; ND, means notdetermined.

The purity of the nonhematopoietic cells was demonstrated by a nearcomplete absence of two macrophage markers, CD68 and cathepsin B (asshown by Northern blotting data, FIGS. 6A and 6B). As a positivecontrol, bone marrow mononuclear cells rich in myelomonocytic cellsabundantly expressed CD68 (lanes 5 & 6, FIG. 6A). The Northern blotresults are consistent with a purity estimate of ˜95% (vs. 60% inunfractionated samples) based on morphology and immunocytochemicalstaining for CD68.

Compared to unfractionated cells, the purified nonhematopoietic cellsexpressed significantly higher levels of markers representing fat cells(adipsin, FIG. 6D); osteoblasts (osteoblast-specific cadherin-11,chondroitin sulfate, collagen type 1 and decorin, FIGS. 6E-H);fibroblasts (fibronectin, FIG. 6J); and smooth muscle cells (caldesmonand transgelin, FIGS. 6K-L). No trace of osteoblast, fibroblast, orsmooth muscle cell markers were detected in the bone marrow mononuclearcells, suggesting a less than detectable level of stromal cells or theirprecursors in bone marrow mononuclear cells. However, the fat cellmarker, adipsin, was detected in all samples including the bone marrowmononuclear cells.

Taken together, the morphologic, cytochemical and immunocytochemicalresults (FIGS. 4A-H and Table 1), and the Northern blotting data (FIGS.6A-M) indicate that the nonhematopoietic stromal cells of the Dextercultures co-express markers specific for at least four differentmesenchymal cell lineages.

This finding is especially intriguing because pluri-differentiation isoften a feature of neoplastic cells (Brambilia and Brambilia, Rev. Mal.Respir. 3,235 (1986); Pfeifer et al., Cancer Res. 51, 3793-3801 (1991);Tolmay et al., Virchow's Arch 430, 209-12 (1997). However, a cytogeneticanalysis of the Percoll-gradient purified MPCs showed a normal GTWbanding pattern.

RT-PCR Analysis for Expression of Representative Hematopoietic GrowthFactors and Extracellular Matrix Receptors by MPCs: RT-PCR was conductedin a total reaction volume of 100 μl using 2 μg each of total RNA;corresponding primers; and a master mix of the PCR reagents. The RTconditions included sequential incubations at 42° C. for 15 minutes, 99°C. for five minutes, and 5° C. for five minutes. The PCR conditionsincluded: initial melting at 94° C. for four minutes; and cyclicalmelting at 94° C. for 45 seconds, annealing at 55° C. for 45 seconds andextension at 72° C. for 45 seconds with 34 cycles. PCR was terminatedafter final extension at 72° C. for ten minutes. Reaction products(G-CSF, SCF, each 25 μl; VCAM-1, ALCAM, each 50 μl; ICAM-1, 75 μl) wereconcentrated as necessary; electrophoresed along with a 100-bp DNAladder (GIBCO-BRL) in a standard agarose (1%) gel in TAE buffer; andstained with ethidium bromide.

PCR products, shown in FIG. 7 lanes labeled 1-2, were generated usingaliquots of the same RNA samples from purified stromal MPCs, as used forNorthern blotting shown under FIG. 6 lanes 3 and 4, respectively. Thegene transcripts amplified were as follows: G-CSF (granulocyte-colonystimulating factor); (Tachibana et al., Br. J. Cancer, 76, 163-74(1997); SCF (stem cell factor, i.e., c-Kit ligand); (Saito et al.,Biochem, Biophys. Res. Commun., 13, 1762-69 (1994); ICAM-1(intercellular adhesion molecule-1, CD54) and VCAM-1 (vascular celladhesion molecule-1, CD106) (primers from R&D); and ALCAM (activatedleukocyte cell adhesion molecule, CD166) (Bruder et al., J. Bone Miner.Res., 13, 655-63 (1998)).

The observed PCR products for G-CSF (600 bp, i.e., the top bright band)and ALCAM (175 bp) were significantly different from the expected sizes(278 bp; 372 bp, respectively). However, sequencing of the gel-purifiedPCR bands and subsequent BLAST search showed a 99-100% identity withrespective members. Attempts to detect c-Kit (i.e., SCF receptor) usingprimers as described (Saito et al., Biochem, Biophys. Res. Commun., 13,1762-69 (1994)) amplified a PCR product of ˜300 bp with no homology toc-Kit (data not shown). The observed product sizes for SCF (.about.730bp); ICAM-1 (.about.750 bp); and VCAM-1 (.about.500 bp) were asexpected.

As illustrated in FIG. 7, RT-PCR analysis showed that purified,multi-differentiated MPCs express both critical hematopoietic growthfactor/cytokines, such as G-CSF and SCF as well as matrixreceptors/hematopoietic cell adhesion molecules, i.e. ICAM-1, VCAM-1,and ALCAM.

Example 2 Comparison of the Ability to Support In Vitro Hematopoiesis byPurified MPCs vs. Unfractionated Bone Marrow Stromal Cells

CD34+ positive cells (hematopoietic progenitor cells) were purified(Dynal kit) and cocultured with irradiated stromal monolayers for fiveweeks, followed by performance of standard colony assays forhematopoietic progenitors using methylcellulose medium supplemented withcolony stimulating factors (using MethoCult medium from Stem CellTechnologies, Inc, Canada). Unfractionated bone marrow stromal cells andpurified MPCs were prepared in the same manner as in Example 1. Data inFIG. 8 represents results from three experiments. Purified MPC providesincreased preservation of hematopoietic progenitor cells compared tounfractionated stromal cells.

Example 3 Animal Model for Enhanced Engraftment Capacity of MPCs

The Severe Combined Immunodeficiency Disease (SCID) mouse model is anideal system in which to investigate MPC function. Engraftment of humanhematopoietic progenitors in SCID mice requires either coadministrationof exogenous human cytokines, or cotransplantation of human bone marrowplugs or bone fragments.

There has been discovered a convenient, new source for human bone marrowstromal cells for enhancing transplantation that does not requirecytokines, bone fragment, or marrow. Unlike prior methods, the isolatedcells of the present invention support human hematopoiesis in the SCIDmouse model as effectively as whole marrow stroma. The transplantationof human marrow mononuclear cells combined with purified MPCs results indramatically vigorous engraftment of human cells in spleen, bone marrow,liver, pancreas, lungs, stomach, and paravertebral neuronal ganglia ofSCID mice. By contrast, mice receiving human bone marrow mononuclearcells alone or MPCs alone expectedly showed no detectable evidence ofhuman hematopoietic cell engraftment. Also notably, the mortality ratewas highest in mice that received unfractionated whole marrow stromawhereas purified MPC increased the survival rate which can be due toreduction in GvHD.

Transplantation of Human Cells in SCID Mice: Homozygous CB-17 scid/scidmice, six to eight weeks of age, were used. Lyophilized anti-asialo GM1rabbit antibody (Wako Chemicals) was suspended in 1 ml sterile ddH₂O,followed by pretreatment of mice with an IP injection of 20 ml (600 mg)ASGM1 antibody (to specifically deplete mouse macrophages and NK cells).Alternatively, one could use NOD/SCID mice lacking NK cell function,however, in light of highly promising preliminary results it was electedto continue use of scid/scid mice. The antibody treatment scheduleincluded four-hour pre-engraftment and every seven days thereafter forthe duration of the experiment. On the day of transplantation, the micewere irradiated with 200 or 300 cGy gamma-irradiation from a ¹³⁷Cssource. Approximately 2.5×10⁶ MPCs suspended in 0.5 ml McCoy's mediumand/or 25×10⁶ MNCs suspended in 0.2 ml were injected per mouse,intraperitoneally. Hematopoietic cell engraftment was assessed afterfive weeks by harvesting and analyzing representative hematopoietic andnonhematopoietic organs including blood, spleen, bone marrow (from twofemurs and tibia) from euthanized mice.

Flow Cytometric Evidence: FIGS. 9A and 9B are flow cytometric evidenceof human hemopoietic cells in a SCID mouse cotransplanted with marrowMPC. FIG. 9A shows the presence of CD45+/CD34+ progenitors in themarrow. FIG. 9B shows CD45/CD34-mature hematopoietic cells circulatingin the mouse's blood.

Photomicrographs of Cells: FIGS. 10A-H shows engraftment of humanhematopoietic cells in a SCID mouse cotransplanted with the purifiedmarrow MPCs of the present invention. FIG. 10A shows a serial section ofa mouse spleen stained with H & E. FIG. 10B shows a serial section of amouse spleen stained with immunoperoxidase stain for CD45. FIG. 10Cshows bone marrow stained for CD45. FIG. 10D shows a serial section ofthe mouse liver stained with H&E depicting involvement of periportalareas. FIG. 10E shows a serial section of the mouse stomach stained withH&E showing transmural infiltration. FIG. 10F shows a serial section ofthe mouse lung stained with H&E showing involvement of peribronchialarea. FIG. 10G shows a serial section of the mouse pancreas stained withH&E. FIG. 10H shows a serial section of the mouse paravertebral gangliastained with H&E.

FIG. 11A is a photomicrograph of a serial section of the spleen of anormal BALB/C mouse showing white pulp populated by darkly staininglymphocytes (H&E). FIG. 11B is a photomicrograph of the spleen of a SCIDmouse showing white pulp largely consisting of lightly staining stromalframework (H&E). FIG. 11C is a photomicrograph of the spleen of a SCIDmouse cotransplanted with human bone marrow MNC and the purified bonemarrow MPCs of the present invention showing homing (engraftment) ofhuman B cells to white pulp.

Southern Blotting Data: Hybridization of sample DNA using a DNA probespecific for human chromosome 17 alpha satellite DNA (p17H8) showslinear signal intensity with a 2.7 Kb band (arrow; autoradiogram exposedfor only 45 minutes) (FIG. 12A). Lanes 1-10 contain human DNA starting1000 ng to 100 ng admixed with 0 ng 900 ng of mouse DNA, total amountDNA loaded in each lane being 1 ug, allowing construction of a standardcurve. The reported limit of detection with this technique is 0.05%human cells, which is more reliable than flow cytometry in detectingvery low levels of human cell engraftment.

FIG. 12B is a Southern blot of EcoR1 digest of thymic genomic DNA fromSCID mice. Lanes 1-5 were loaded with 500 through 100 ng human DNA.Lanes 6, 9-11 were loaded with DNA from mice which receivedunfractionated bone marrow stroma plus bone marrow mononuclear cells.Lanes 7, 8, 14, 15 were loaded with DNA from mice that received MPCsplus bone marrow mononuclear cells. Lanes 12, 13. were loaded with DNAfrom mice that received bone marrow mononuclear cells only. There isevidence of human cell engraftment in the mouse thymus in lanes 9 and 11and lanes 14 and 15 evidenced by the 2.7 Kb band. There was no evidenceof engraftment in mice that only received only bone marrow mononuclearcells, lanes 12 and 13.

FIG. 12C is EcoR1 digest of Lymph Node genomic DNA from SCID mice. Lanes1-5 were loaded with 500 through 100 ng human DNA. Lanes 6, 9-11 wereloaded with DNA from mice which received unfractionated bone marrowstroma plus bone marrow mononuclear cells. Lanes 7, 8, 14, 15 wereloaded with DNA from mice that received MPCs plus bone marrowmononuclear cells. Lanes 12, 13 were loaded with DNA from mice thatreceived bone marrow mononuclear cells only. While there was evidence ofengraftment of human cells in the mouse lymph nodes for mice thatreceived unfractioned bone marrow stromal cells and MPCs, there was noevidence of engraftment in mice that only received only bone marrowmononuclear cells, lanes 12 and 13.

Increased Survival and Evidence of MPC Effect on GvHD: FIGS. 13A-1,13A-2, 13B-1, and 13B-2 show graphs comparing the survival rate andengraftment of human hematopoietic cells in SCID mice cotransplantedwith the purified bone marrow MPCs of the present invention versusunpurified bone marrow stromal cells. Mice in FIGS. 13A-1 and 13A-2received 300 cGy irradiation dose and mice in FIGS. 13B-1 and 13B-2received 200 cGY of irradiation. FIGS. 13A-1, 13A-2, 13B-1, and 13B-2show comparable engraftment of human hematopoietic cells in SCID micecotransplanted with purified MPCs versus unpurified bone marrow stromalcells and the markedly enhanced survival of mice receiving purifiedMPCs. Notably, no engraftment was observed in mice receiving bone marrowmononuclear cells alone.

The highest mortality rate, FIGS. 13B-1 and 13B-2, was observed in micereceiving the unpurified stromal cells and the bone marrow mononuclearcells. The increased mortality observed can be related to the presenceof highly immunogenic macrophages and consequent GvHD. The mice with thehighest survival rate, as shown in FIGS. 13A-1 and 13A-2, were the micereceiving purified MPCs and bone marrow mononuclear cells.

FIGS. 14A-D demonstrate apoptosis by TUNEL assay in organs of SCID micethat died after transplantation with human bone marrow mononuclear cellsand unpurified bone marrow stromal cells. FIG. 14A shows a serialsection of the liver of the mouse that survived. FIG. 14B shows a serialsection of the liver of the mouse that died. FIG. 14C shows a serialsection of the spleen of the mouse that survived. FIG. 14D shows aserial section of the spleen of the mouse that died. Hematoxylincounterstain was applied to sections in FIG. 14A and FIG. 14C.Methylgreen counterstain was applied to sections in FIG. 14B and FIG.14D.

Notably, there is discrete TUNEL-positive nuclei in the liver of theexpired mouse in FIG. 14B and complete absence of staining in the liverof the surviving mouse FIG. 14A. While some ill-defined globules ofstaining are observed in the spleen of the mouse that survived, thenuclear integrity of most of the cells is well preserved suggestingminimal or no apoptosis (FIG. 14C). By contrast, the dead mouse spleen(FIG. 14D) showed extensive TUNEL positivity precluding accurateinterpretation. Control mouse liver and spleen showed results similar tothose of the mouse that survived.

The size of the spleens from the mice that survived and the mice thatdied were compared. The dead mice were observed to have small andatrophic spleens correlating with lymphoid cell depletion and apoptosis.

The above results indicate that purified MPC can support humanhematopoiesis in SCID mice as effectively as whole marrow stroma.Equally important is that the purified MPCs increased the survival rate.Evidence suggests that the increased survival can be due to a reductionin GVHD.

Example 4 Administration of Bone Marrow Cells and Mesenchymal ProgenitorCells to Breast Cancer Patients Treated with Chemotherapy

A breast cancer patient undergoes a diagnostic posterior iliac crestbone marrow aspiration and biopsy using a local anesthetic. A smallportion (2 to 3 ml) of the aliquot (10 to 20 ml) of marrow is submittedfor routine histologic testing and determination of the presence oftumor cells using immunoperoxidase testing. The remainder of the cellsare Dexter cultured for MPCs as described above in Example 1.

The patient also undergoes placement of a pheresis central venouscatheter, and receives subcutaneous injections of G-CSF (filgrastin) 10μg/kg/day as described in Peters, et al, Blood, Vol. 81, pgs. 1709-1719(1993); Chao, et al, Blood, Vol. 81, pgs. 2031-2035 (1993); Sheridan, etal, The Lancet, Vol. 2, pgs. 891-895 (1989); and Winter, et al, Blood,Vol. 82, pg. 293a (1993). G-CSF injections begin at least three daysbefore the first pheresis is initiated. G-CSF therapy is withheld if thewhite blood cell count rises above 40,000/μL and is resumed once thewhite blood cell count drops to less than 20,000/μL.

If the patient is receiving only G-CSF as the vehicle for “mobilization”of peripheral blood progenitor cells, the patient must not have receivedchemotherapy within four weeks of the planned pheresis. If the patienthas received both conventional chemotherapy and G-CSF treatment formobilization, the patient must not have received chemotherapy within tendays of the planned pheresis, and the white blood cell count must be atleast 800/μL and the platelet count at least 30,000/μL.

Daily pheresis procedures are performed using a Cobe Spectra instrument(Cobe, Lakewood, Col.), and each cellular collection is cryopreservedusing a controlled-rate liquid nitrogen freezer, until at least 15×10⁸mononuclear cells/kg are collected (Lazarus, et al., Bone MarrowTransplant, Vol. 7, pgs. 241-246 (1991)). Each peripheral bloodprogenitor cell is processed and cryopreserved according to previouslypublished techniques. (Lazarus, et al., J. Clin, Oncol., Vol. 10, pgs,1682-1689) (1992); Lazarus et al., (1991)).

Eight days before the patient is infused with the autologous peripheralblood progenitor cells, the patient receives chemotherapy over a periodof 96 hours (four days), with the following chemotherapy agents: 1)Cyclophosphamide in a total dosage of 6 g/m² (1.5 g/m²/day for fourdays) is given via continuous intravenous infusion at 500 mg/m² in 1,000ml normal saline every eight hours; 2) Thiotepa in a total dosage of 500mg/m²/day for four days) is given via continuous intravenous infusion at125 mg/m² in 1,000 ml normal saline every 24 hours; and 3) Carboplatinin a total dosage of 1,800 mg/m² (200 mg/m²/day for four days) is givenvia continuous intravenous infusion at 200 mg/m² in 1,000 ml of 5%dextrose in water every 24 hours.

The patient also receives 500 mg of Mesna in 50 ml normal saline IV over15 minutes every four hours for six days (144 hours), beginning with thefirst dose of cyclophosphamide.

At least 72 hours after the completion of the chemotherapy, the MPCs areharvested from the Dexter culture(s). MPCs are collected and purified asdescribed in Example 1. Cells are resuspended at approximately 10⁶cells/ml, and injected slowly intravenously over 15 minutes to provide atotal dosage of from 10 to about 5×10⁶ cells.

MPCs can also be frozen and thawed to use when needed. For example,unfractionated cells from a Dexter culture are frozen. Upon thawing thecells are plated for about two days. The MPCs are then purified as inExample 1 above. The MPCs are then replated with serum or in a serumfree media and can remain stable for up to six days.

The day after the patient receives the MPCs, the frozen autologousperipheral blood progenitor cells are removed from the liquid nitrogenrefrigerator, transported to the patient in liquid nitrogen, submersedin a 37° C. to 40° C. sterile water bath, and infused rapidlyintravenously without additional filtering or washing steps. GM-CSF inan amount of 250 μg/m² then is given as a daily subcutaneous injection,beginning three hours after completion of the autologous bloodprogenitor cell infusion. The GM-CSF is given daily until the peripheralblood neutrophil count exceeds 1,000/μL for three consecutive days.

Example 5 Genomic Changes Observed in Leukemia Associated MPCs

The following is one example of how normal hematopoiesis might becompromised in leukemic conditions. The cellular interactions thatunderlie leukemic bone marrow involve stromal cells, leukemia/lymphomacells, and normal hematopoietic pro genitors (including those ofmyelopoiesis, erythropoiesis and megakaryocytopoiesis). In addition todisplacing normal hematopoietic cells, the leukemia/lymphoma cells canpotentially cause direct damage to the hematopoietic supportive stromalcells by inducing unwanted gene expression profiles and adverselyaffecting the normal hematopoiesis. The cellular interactions can beschematized as:

The point of this scheme is that regardless of whether stromal celllesions are primary or secondary to leukemogenesis, the normalhematopoietic function is invariably compromised in leukemic conditions,though different leukemias affect myelopoiesis, erythropoiesis andmegakaryocytopoiesis differentially. Contrary to the prevailing notion(see Marini, F et al., Mesenchymal Stem Cells from Patients with ChronicMyelogenous Leukemia Patients can be Transduced with Common GeneTransfer Vectors at High Efficiency, and are Genotypically Normal, 42ndAnnual Meeting of the American Society of Hematology, Dec. 1-5, 2000Poster #665), there has been observed extensive and striking geneexpression changes in leukemia-associated bone marrow MPCs by usinghigh-resolution genomics. Therefore, one embodiment of the presentinvention is to use transplantation of tissue-culture expanded, purifiednormal MPCs to improve granulopoiesis, erythropoiesis andthrombopoiesis, in for example MDS (most of MDS patients do not die fromblast transformation but from complications related to cytopenias, i.e.,hematopoietic failure).

The studies targeted acute myeloid leukemia (AML), chronic myeloidleukemia (CML) and multiple myeloma (MM), one case of each. The AMLpatient was a 57 year-old woman with 52% myeloblasts in the bone marrowwith immunophenotype confirmed by flow cytometry and a karyotypicabnormality of 45, XX, −7(6)146, XX [6]. Together with morphology, thediagnosis was AML arising in a background of myelodysplasia. The CMLpatient was a 35 year-old man with 2% blasts in the bone marrow andkaryotypic abnormalities of Philadelphia chromosome and BCR/ABL generearrangement. Together with morphology, the diagnosis was CML inchronic phase. The MM patient was a 61 year-old woman with a IgAmyeloma. The serum IgA level was 2.4 g/dl and the marrow plasma cellcount was 37%. None of the patients was treated prior to obtainingmarrow samples used in this study, to avoid any therapy-induced changescomplicating the disease-associated changes.

The leukemic samples consisted of marrow aspirates that remained unusedafter clinical diagnostic studies were preformed. A bone marrow sampleobtained from an adult healthy male who had consented to donate bonemarrow for standard marrow transplantation was simultaneously studied.The normal bone marrow sample consisted of residual cells recovered fromthe filters after complete filtration of the marrow sample. Setting upof Dexter-type stromal cell cultures and isolation of MPC were asdescribed in Example 1. The normal stromal cells were studied withoutand after stimulation with TNFα because TNFα (and IL-4) are regarded asnegative regulators of hematopoiesis. Notably these cytokines,especially TNFα, are elevated in marrow plasma of patients withmyelodysplastic syndromes (MDS), the clinical hallmarks of which areanemia, leukopenia and thrombocytopenia (i.e., pancytopenia). TNFα andIL-4 are considered possible mediators of hematopoietic dysregulationtypical of MDS.

A Stepwise Genomics Strategy Encompassed: Preparation of total RNA fromMPC samples→generation of cDNA→preparation of ds DNA→in vitrotranscription into cRNA→fragmentation of cRNA→hybridization of targetRNA to a microarray of known genes (Affymetrix genechip containing DNAfrom ˜12,000 known human genes, e.g., U95A oligonucleotidemicroarray)→analysis of differentially expressed genes using anappropriate software (GENESPRING) to discern the patterns of geneexpression or genomic signatures by a given MPC type.

Cluster Analysis Showing Gene Expression Patterns in Bone Marrow MPCIsolated from a Normal Individual and Patients with Different LeukemicConditions: Genes with correlated expression across bone marrow MPCtypes: GENESPRING was used for cluster analysis. Prior to application ofan agglomerative hierarchical clustering algorithm, microarray signalswere normalized across experiments (i.e., from one MPC type to another)making the median value of all of measurements unity, so differentexperiments are comparable to one another. The signals were alsonormalized across genes in order to remove the differing intensitysignals from multiple experimental readings. Genes that are inactiveacross all samples were eliminated from analysis. Notably, 7398 genesout of 12,626 genes (present on the Affymetrix genechip used) passed thefilter of a normalized signal intensity of at least 0.1 across at leastone of the five experiments performed. Cluster analysis was performedwith standard correlation (same as Pearson correlation around zero) asthe distance metric, a separation ratio of 0.5 and a minimum distance of0.001 as provided by the software application. A closer relationshipbetween CML- and MM-associated MPCs was observed, which in turn arerelated to AML-associated MPC, thus transforming global patterns of geneexpression into potentially meaningful relationships.

Two-dimensional cluster analysis of tissue vs. gene expression vectors:A gene tree was constructed. Genes cluster near each other on the “genetree” if they exhibit a strong correlation across MPC experiments andMPC tree branches move near each other if they exhibit a similar geneexpression profile. The data indicated that the two-way clusteringreadjusted the location of a number of genes resulting in accentuationof genomic signatures of each cell type. Investigators can usefullycatalog genes composing any unique or signature cluster of interest bycreating a gene list and disclosing their identities.

Self-Organizing Map (SOM) Clusters (6×5) Show Differential GeneExpression in Bone Marrow MPC Isolated from Different HematopoieticConditions: Generation of SOM clusters involved prior normalization andfiltering of the data. SOM algorithm was applied as provided byGENESPRING. Visualization of SOM clusters in combination withhierarchical clustering (i.e. MPC tree) revealed correlated meaningfulpatterns of gene expression. Predicated on the basis of SOM operatingprinciple, the related SOM clusters tend to be located physically closeto each other. For example, the juxtaposition of the SOM clusters withthe common denominator containing genes that are up-regulated inAML/MDS-associated MPC. Whole or part of any SOM cluster can be selectedto make a gene list providing the identities of the genes involved.

Genes Highly Expressed in Normal MPC but Absent or Minimally Expressedin Leukemia-Associated MPC: Lists of genes that are down-regulated inleukemia-associated MPC (AML/MDS, CML and MM) were created in comparisonto normal MPC. A Venn diagram was made using these three gene lists.GENESPRING allows creation of sublists of genes corresponding to union,intersection and exclusion. Transcriptional profiles of any of thesesublists of genes can be visualized across MPC samples of interest. Thefollowing is one such sublist of genes containing genes that are highlyexpressed in normal MPC and down-regulated in leukemia-associated MPCsrevealing the identity of the subset of genes of interest: putative,wg66h09.x1 Soares Homo sapiens cDNA clone, Homo sapiens mRNA forCMP-N-acetylneuraminic acid hydroxylase, Homo sapiens cDNA cloneDKFZp586G0421 (symptom: hute 1), Human mRNA for histone H1x, Putativemonocarboxylate transporter Homo sapiens gene for LD78 alpha precursor,Interacts with SH3 proteins; similar to c-cbl proto-oncogene product,wg82b12.x1 Soares Homo sapiens cDNA clone, Human atrial natriureticpeptide clearance receptor (ANP C-receptor) mRNA, Human 71 kDa 2′5′oligoadenylate synthetase (p69 2-5A synthetase) mRNA, Homo sapienshMmTRA1b mRNA, Human GOS2 protein gene, Preproenkephalin, Humanguanylate binding protein isoform I (GBP-2) mRNA, Human gene forhepatitis C associated microtubular aggregate protein p44, 17-kDaprotein, Human insulin-like growth factor binding protein 5 (IGFBP5)mRNA, GS3686, Human monoamine oxidase B (MAOB) mRNA, Insulin-like growthfactor II precursor, Human insulin-like growth factor binding protein 5(IGFBP5) mRNA, Similar to ribosomal protein L21, X-linked mentalretardation candidate gene, and Homo sapiens mRNA; cDNA DKFZp434A202.

Genes not expressed in normal MPC but highly expressed inleukemia-associated MPC: Lists of genes that are upregulated (instead ofdown-regulated) in leukemia-associated MPCs (AML/MDS, CML and MM) werecreated in comparison to normal MPC and a Venn diagram was made. Thefollowing is one such sublist of genes containing genes that areinactive in normal MPC but up-regulated in leukemia-associated MPCsrevealing the identity of the subset of genes of interest:Beta-tropomyosin, Homo sapiens clone 24659 mRNA sequence, Human mRNA forDNA helicase Q1, OSF; contains SH3 domain and ankyrin repeat, ym22b12.r1Soares infant brain INIB Homo sapiens cDNA clone, Human mRNA forpre-mRNA splicing factor SRp20, Human mRNA for golgialpha-mannosidasell, OSF-2os, Homo sapiens gene for Proline synthetase,hk02952 cDNA clone for KIAA0683, wi24g10.x1 Homo sapiens cDNA clone,Lysosomal enzyme; deficient in Sanfilippo B syndrome, CTP synthetase (AA1-591), WD repeat protein; similar to petunia AN11, Human mRNA for5′-terminal region of UMK, complete cds, Homo sapiens chemokine exodus-1mRNA, complete cds, Human GPI-H mRNA, complete cds, Homo sapiens mRNAencoding RAMP1 Transforming growth factor-beta-2 precursor, and Homosapiens mRNA for KIAA0763 protein.

Visualizing Expression of Phenotypically & Functionally Relevant Genesacross Samples of Normal & Disease-Associated BM MPC: AlthoughGENESPRING is a highly flexible and user-friendly software application,it lacks the facility to create functionally relevant gene listscontaining user-defined key words. This limitation was overcome bydevising the following method via MICROSOFT EXCEL. A stepwise protocolto create such a gene list using EXCEL includes: Open the annotatedmicroarray genome file (e.g., Affymetrix U95A) in EXCELS select thecolumn with gene names→select Data from pull-downmenu→Filter→AutoFilter→Custom→enter key words (e.g., cell adhesion orcell cycle)→OK→generates a new EXCEL worksheet with the list of genescontaining the key words. Copy and paste the list of genes containingthe key words into GENESPRING and save the gene list with a meaningfulname. Twenty-two (22) such functionally relevant gene lists (Table 2)were created.

The resulting approach is a simple and powerful way to peer into theexpression profiles of focused sets of functionally relevant genesacross samples of interest. For example, the human vascular celladhesion molecule-1 (VCAM-1) gene is completely down-regulated inAML/MDS and the human insulin-like growth factor binding protein(hIGFBP1) gene is up-regulated in AML compared to all other samples.Similarly, Homo sapiens gene for LD78 alpha precursor is down-regulatedin all of leukemia-associated MPCs. Finally, the lineage markers CD45and CD68 are essentially absent from the leukemia-associated MPCsattesting to the high degree of purity achieved by the samplepreparation technique of the present invention.

Results: The genomic changes observed in leukemia-associated MPCs arestriking. As shown in Table 2, the changes (up-regulation and/ordown-regulation) involved hundreds of genes. These changes were mostdramatic in MPC associated with AML arising in a background of MDS andinvolved multiple classes of genes (Tables 1-2). Expectedly, theTNFa-induced changes were extensive. Given the high level of purity ofMPC preparations, the enormous genomic changes observed are reflectiveof the underlying pathologic lesions in the MPCs themselves (and not dueto the contaminating leukemic cells and/or macrophages). These studiesstrongly support the hypothesis that stromal cells in a leukemic patientare functionally defective and therefore purified MPCs are of value inrestoring the loss of hematopoietic function in leukemic patients.

TABLE 2 Magnitude of global gene expression changes inleukemia-associated and TNFa-stimulated MPCs in comparison to normal MPCAML/MDS MPC CML MPC MM MPC TNFa MPC # of genes up- 234 112 108 279regulated # of genes 379 208 251 164 down-regulated

TABLE 3 Functional classes of genes analyzed across normal andleukemia-associated MPCs Annexins (14) Caspases & apoptosis-relatedtranscripts (33) Cadherins (50) Calmodulins/calmodulin- dependentkinases (25) Cell adhesion molecules (20) Cathepsins (19) Collagens (71)Cell division cycle-related transcripts (36) Cytokines (19) Epidermalgrowth factors and related transcripts (22) Fibroblast growth factors(21) Fibronectins (6) Galectins (6) Growth factors (136) IGF system (24)Interleukins/receptors (76) Integrins/disintegrins (70) Lineage-relatedmarkers (19) Laminins (13) Platelet-derived growth factors & receptors(12) TNF alpha-related transcripts (29) TGF beta-related transcripts(25)

The gene lists in Table 3 were created as described above and analyzedusing GENESPRING. The numerical value in parenthesis refers to thenumber of transcripts in the corresponding class of genes analyzed.

Example 6

The present invention provides the following benefits: a) identificationand documentation of BM stromal cell gene expression patterns undervaried, normal, and leukemic hematopoietic conditions; b) identificationof stromal cell genes that can be therapeutic targets for improvement ofnormal hematopoietic function that is constantly compromised in leukemicpatients, and identification of similar targets for arresting the growthand progression of neoplastic clones since stromal cells provide thenecessary support for preferential growth of leukemic cells (CLL, MM)within BM and protect the leukemic cells from chemotherapy-induced death(MM); and c) identification of new biological bases and new diagnosticmarkers for refinement of the classification and diagnosis of leukemia.This present invention can also lead to important insights into thepathogenesis of leukemia. In broad terms, analysis of global geneexpression or transcriptome (transcriptional profile composed of alltranscribed regions of the genome) is considered a nonbiaseddiscovery-driven (as opposed to hypothesis-driven) approach to theanalysis of gene expression. A stepwise genomic strategy encompassespreparation of total RNA from cells of interest, to generation of cDNA,to preparation of ds DNA, to in vitro transcription into cRNA, tofragmentation of cRNA, to hybridization of target RNA, to a microarrayof known genes (and/or ESTS), to analysis of differentially expressedgenes using an appropriate software to discern the patterns of geneexpression or genomic signatures by a given disease-associated celltype.

Furthermore, the present invention can test the utility of samplepreparation technology applied to normal EM-derived MPCs (untreated andtreated with representative cytokines) and MPCs derived from patientswith representative pre-leukemic and leukemic conditions for performanceof high-resolution DNA microarray technology (Affymetrix genechipcontaining DNA from 12,000 known human genes, e.g. U95A oligonucleotidemicroarray).

Representative cytokines which are pathologically altered inhematopoietic conditions and that can be used in this study includeTNF-α, TGF-β and interferon-γ. The pre-leukemic conditions includemyelodysplastic syndromes (MDS) and the leukemic conditions includechronic myeloid leukemia (CML), acute myeloid leukemia (AML), acutelymphocytic leukemia (ALL), and multiple myeloma (MM).

The front-end strategy of microarray analysis involves the use ofPercoll-gradient purified MPCs. As a follow-up strategy, to validate thestromal cell origin of the differentially expressed genes, MPCs obtainedfrom cytospins of BM stromal cells by laser-capture microdissection(LCM) selected on the basis of morphology (FIG. 3) are used followed by“real-time” quantitative polymerase chain reaction (PCR). This can beperformed with an LGM system as well as a “real-time” QPCR system.Validation can be performed on at least one sample from each of 6 normalBM M7NC/MPC types and on one sample from each of 5 leukemia-associatedMPC types. Validation is considered successful if the microarray resultsand PCR results on a given MPC sample match using a suite of 20 genesselected based on median pattern of microarray results for the givencell type. This approach not only validates the microarray results butalso ascertains the stromal cell origin of the expressed genes.

The standard published protocols involving LGM and “real-time”quantitative PCR and the instructions accompanying the equipment areused for performing the experiments.

Stepwise LCMJ real-time QPCR protocol entails the following. Cytospinsare made from BM stromal cells. The cytospins are stained withhematoxylin and MPC is selected for based on morphology. Microdissect upto 1,000 MPC from each sample. RNA is extracted and reverse transcribedinto cDNA. The cDNA is amplified using gene-specific primers and“real-time” quantitative PCR.

By applying the combined power of different analytical techniques (suchas hierarchical clustering and self-organizing maps) together with therecently developed sample preparation technology for stromal cells thepresent invention provides a molecular biological basis that can allowrefinement of the classification and diagnosis of leukemias andlymphomas, uncovering the suspected disease heterogeneity. This enablesthe deciphering of the genomic expression profiles or signatures of bonemarrow stromal cells in about 10 different physiologic states and about20 different leukemic states. In addition to aiding in refinement of theclassification and diagnosis of the hematopoietic malignancies, the dataprovides clues to potential novel drug targets and insights intopathogenesis.

The present invention functions by identifying the MPC genes that aredifferentially expressed after stimulation with different hematopoieticcytokines implicated in the pathogenesis of pre-leukemic conditions(MDS); in actual pre-leukemic disorders (MDS); and in overt leukemias(CML, AML, CLL, ALL, MM) as well as in lymphomas that have a leukemicphase with involvement of BM.

The present invention is accomplished by first determining the mediangene expression profiles for MPCs associated with each disease andstimulated by each cytokine of interest (this objective can be achievedby treating the gene expression vectors of individual cases in each MPCcategory as replicates; this capability is available in GENESPRINGsoftware application). Then the gene groups that are up regulated anddown regulated and that are common to all the members in a given MPCcategory are identified (this is accomplished using a series of Venndiagrams and creating required gene lists via GENESPRING). Finally, theup-regulated and down-regulated gene sets for a given disease-associatedor cytokine-stimulated MPC are combined. This allows the identificationof gene sets with minimal number of elements that are unique to a givenMPC type with a capability to discriminate one MPC type from another(this can also be accomplished by means of a series of Venn diagrams andlists of required genes obtained via GENESPRING). Such gene sets can beof immense diagnostic value as they can be routinely used in an assaysimpler than microarray analysis (for example “real-time” quantitativePCR). Such gene sets can additionally provide insights into pathogenesisand possible targets for design of new drugs.

Determine expression profiles of MPC genes which are regulated as aresult of exposure of normal MPCs to cytokines that are known to have ahematopoietic support role and/or are abnormally elevated inpre-leukemic/leukemic conditions, i.e., TNF α; IL-4; TNF α+IL-4;interferon γ; TGF β; PDGF; FGF; EGF; and calmodulin.

TNF α, IL-4 and IFN γ are potent negative regulators of hematopoiesis.Notably these cytokines, especially TNF α, are elevated in marrow plasmaof patients with myelodysplastic syndromes (MDS), the clinical hallmarksof which are anemia, leukopenia and thrombocytopenia (i.e.,pancytopenia). TNF α and IL-4 are thus possible mediators ofhematopoietic dysregulation typical of MDS. Studies regarding theseregulators can uncover the molecular pathways leading to cytopenias inMDS patients. As indicated earlier, myeloproliferative disorders areanother, in some ways similar, group of hematopoietic disorders that areclonal in origin but not overtly malignant clinically. These MPDsinclude polycythemia vera, essential thrombocythemia, idiopathicmyelofibrosis (agnogenic myeloid metaplasia) and chronic myelogenousleukemia. These disorders have the potential to change from one to theother at any time, however the signals that trigger such conversionremain enigmatic. Idiopathic myelofibrosis (IMF), in which stromal cellsseem to play a profound pathogenetic role, is characterized by fibrosisof the marrow cavity, extramedullary hematopoiesis, splenomegaly, andanemia and leukoerythroblastic features in the peripheral blood. Whilemyeloproliferation is known to be a clonal process, the accompanyingstromal cell proliferation and fibrosis are believed to be a polyclonalreactive process that is likely to be due to increased intramedullaryactivity of a number of cytokines including TGF β, PDGF, FGF, EGF andcalmodulin, as shown by other investigators.

Cancer genomics is a rapidly expanding area of investigation. The focusis unique however in emphasizing not the leukemic cells themselves butrather BM stromal cells that provide a haven to various types ofpre-leukemia and leukemia cells, non-Hodgkin's lymphomas (NHLs) andmetastatic cancers (METs). Pre-leukemic clonal neoplastic conditionsinclude myelodysplastic syndromes (MDSs) and myeloproliferativedisorders (MPD5). Stromal cells are known to produce and/or respond togrowth factors such as EGF, PDGF, FGF, VEGF, and cytokines such as IL-1or TNF a, partially explaining the interactive relationship betweenstromal cells and cancer cells, especially in MDS and CML.

In spite of similarities between BM stroma and non-BM stroma, certainsharp distinctions do stand out. Non-BM stromal cells are terminallydifferentiated fibroblasts, while BM stromal cells represent a uniquepluripotent or pluridifferentiated mesenchymal cell type, thusexhibiting preserved developmental “plasticity”. Using 5-10 cc BMaspirate samples from adult leukemic patients and 3-5 cc BM samples frompediatric patients with ALL, the study can analyze the BM stroma. One ccof marrow sample can produce at least 1 T-150 flask of stromal cells.One concern is that it can be hard to obtain marrow samples from caseslike CML and myelofibrosis. In such cases stromal cells are grown usingperipheral blood samples as described in the prior art. At least oneflask of stromal cells (i.e., 1 cc marrow) to yield the RNA required foranalysis. About 10 cases of each type of leukemia/lymphoma were studied.The study provided important insights into the functioning of the BMmicroenvironment in normal and leukemic hematopoiesis.

A database including all of the above information and that can includeage, gender and associated major illness in terms of clinical/pathologicdiagnosis for each subject/patient can be created. This can also includeinformation on cytogenetic, molecular and flow cytometric studies.Finally, also included can be the information on clinical course interms of disease progression and response to treatment exercisingadequate care to protect the identity of individual patients. The studyanalyzed genomic expression profiles or signatures of bone marrowstromal cells derived from about 12 different normal bone marrow statesand about 19 different leukemia/lymphoma states, approximately 10 casesof each as shown in Table 4, accounting for a total of 310 samples.

Using the information of the present invention, those of skill in theart can: a) study select gene or sets of genes as relevant tohematopoietic disease conditions using relatively inexpensive butlow-throughput technologies such as Northern blotting, RNase protectionassays and/or PCR intended for gene expression analysis; b) reanalyzethe primary data by using newer and more powerful bioinformatic tools asthey become available; and/or c) identify newer drug targets anddiagnostic markers relevant to specific diseases, such as MM or CML etc.

TABLE 4 Scope of human BM samples targeted for DNA microarray analysis(approximately 10 cases of each) Normal BM mononuclear cells (NMNC)Normal BM stromal cells, unfractionated and unstimulated (NBMS) Normalpurified mesenchymal progenitor cells, unstimulated (NMPC) NMPCstimulated with 9 different cytokines: NMPC stimulated with TNFα (TNFαMPC) NMPC stimulated with TGFβ (TGFβ MPC) NMPC stimulated withinterferon y (IFNγ MPC) NMPC stimulated with IL-4 (IL-4 MPC) NMPCstimulated with TNFα + IL-4 (TNFα + IL-4 MPC) NMPC stimulated with PDGF(PDGF MPC) NMPC stimulated with EGF (EGF MPC) NMPC stimulated with FGF(FGF MPC) NMPC stimulated with calmodulin (calmodulin MPC) MDS -Refractory anemia (MDS-RA MPC) MDS - Refractory anemia with ringedsideroblasts (MDS-RARS MPC) MDS - Refractory anemia with excess blasts(MDS-RAEB MPC) MDS - Chronic myelomonocytic leukemia (M1)S-CMML MPC)MPD - Polycytheniia vera (MPD-PV MPC) MPD - Essential thrombocythemia(MPD-ET MPC) MPD - Myelofibrosis (MPD-LMF MPC) CML (CML MPC)AML-M0/M1/M2 (AML-MOJM1JM2 MPC) AML-M3 (APL) (AML-M3 MPC) AML-M4/M5(myelomonocytic) (AML-M4i′M5 MPC) ALL-L1/L2 (lymphoblastic) (ALL-L1IL2MPC) ALL-L3 (Burkitt's) (ALL-L3 MPC) Multiple myeloma (MM MPC) CLLISLL(CLL/SLL MPC) Follicle center cell lymphoma (FCL MPC) Mantle celllymphoma (MCL MPC) Lymphoplasmacytic lymphoma (LPL MPC) Marginal zonelymphoma (MZL MPC).

Human Subjects: This study involves the use of bone marrow (BM) samplesfrom human subjects. BM samples can be obtained from normal subjects(male and female 20-45 years) as well as leukemic patients afterinformed consent is obtained. Leukemic cells can be obtained fromdiagnostic samples of BM of adult and pediatric patients (in those casesin which cells remain unused after clinical diagnostic studies arepreformed; i.e., about 90% of cases).

Example 7

In broad terms, global gene expression analysis is considered anonbiased discovery-driven (as opposed to hypothesis-driven) approach tothe analysis of protein expression. A stepwise proteomics strategyencompasses: solubilization of proteins from cells of interest; 2-D gelelectrophoresis (IPG DALT); staining and image analysis of gels;excision of protein spots of interest; trypsin digestion of proteins;mass spectrometry (MALDI-TOF MS and/or ESI MS/MS) performed on trypticfragments; identification of proteins by database searching. The presentinvention provides a method to analyze the population of expressedproteins (i.e., proteome) of BM MPCs in relation to hematopoiesis incollaboration with a state-of-the-art mass spectrometry facility.

The large-format 2-D gel electrophoretic system is used for reproducibleseparation of MPC proteins and to prepare 2-D PAGE protein maps fornormal bone marrow-derived MPCs (untreated and treated withrepresentative cytokines, e.g., TNF .alpha. and/or IL-4) and for MPCsderived from patients with representative pre-leukemic/premalignant andleukemic/malignant conditions. The pre-leukemic conditions includemyelodysplastic syndromes (MDS) and the leukemic conditions includechronic myeloid leukemia (CML), acute myeloid leukemia (AML), chroniclymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), andmultiple myeloma (MM). The protein samples can consist of culturesupernatants/secreted proteins; extracellular matrix (ECM) proteins;plasma membrane proteins solubilized using a three-step differentialextraction protocol, employing conditions of progressively increasingsolubility; and whole cell lysate proteins similarly solubilized usingthe three-step differential extraction protocol. This subproteomeapproach not only simplifies the 2-D PAGE electrophoretic proteinpatterns but also reveals additional proteins, which would otherwisehave gone undetected.

The system of the present invention can be used to differentiallyexpress MPC proteins (i.e., those that increased or decreased inintensity as compared to 2-D PAGE protein maps of normal, unstimulatedMPCs) using mass spectrometry (MALDI-MS and/or nanoelectrosprayionization MS/MS) and/or Western blotting and/or Western-ligandblotting.

Using high-resolution proteomics with the added power of high-throughputrobotics, enables the system to identify on a larger(semi-comprehensive) scale the MPC proteins that are differentiallyexpressed in conditions that simulate pre-leukemic bone marrow(following stimulation with different cytokines); and in actualpre-leukemic disorders (MDS) as well as in overt leukemias (CML, AML,CLL, ALL, MM).

The system of the present invention enables the identification of MPCproteins whose expression is regulated as a result of exposure of normalMPCs to cytokines that are known to have a hematopoietic support roleand/or are abnormally elevated in pre-leukemic/leukemic conditions,i.e., TNF α; IL-4; TNF α+IL-4; interferon γ, TGF β; PDGF; FGF; EGF; andcalmodulin.

The system of the present invention also enables the identification ofMPC proteins for which expression is altered as a result of exposure ofnormal MPCs to agents that are clinically used for mobilization ofhematopoietic stem cells from BM into peripheral blood to facilitateeasy collection and subsequent transplantation, e.g., G-CSF and G-CSFplus cyclophosphamide.

Further, the system of the present invention enables the identificationof Identify the MPC proteins whose expression is pathologically alteredin hematopoietic disease states such as: MDS, CML, AML, CLL, ALL and MMby matching the 2-D PAGE protein maps of disease-associated MPCs withthe 2-D PAGE database of normal MPCs. If a protein of interest does notexist in the normal MPC proteome, or if it exists in the normal MPCproteome but has not yet been characterized, then such proteins can beidentified by MALDI-MS and/or Nano ESI MS/MS.

The system facilitates understanding of the pathogenetic mechanisms byidentifying the phosphoproteins involved in cell signaling pathways. Thesystems immunoblots the whole cell lysate proteins of normal MPCs,untreated and treated with respective cytokines, using antibodies tophosphotyrosine, phosphoserine, and phosphothreonine. The system thenlocates the corresponding putative phosphoprotein spots on the gel andidentifies the proteins by MALDI-MS and/or Nano ESI MS/MS. Similarly,the system can locate the altered phosphoproteins by immunoblotting thewhole cell lysate proteins of untreated MPCs derived from leukemicpatients. If a protein of interest does not exist in the normal MPCproteome, or if it exists in the normal MPC proteome but has not yetbeen identified, then MALDI-MS and/or Nano ESI MS/MS can identify theprotein.

Bone Marrow MPCs Derived from a Leukemia Background ExpressDistinctively Different Patterns of Cell Adhesion Molecules from NormalMPCs: BM stromal cells provide the background required for homing andsubsequent proliferation and differentiation of hematopoietic stemcells. BM stromal cells also provide a rich microenvironment formetastases and growth of various leukemias. Based on the hypothesis thathoming of normal hematopoietic cells and leukemic cells to marrowutilize the same adhesion mechanisms, it was questioned whether thereare fine regulatory distinctions in terms of quantitative differences inthe expression of the adhesion molecules in normal vs. leukemic BMmicroenvironments. In a pilot study 11 cell adhesion molecules (CAMs)and several lineage-associated markers for Northern blot analysis weretargeted. Dexter-type cultures were grown under standard stromal cellculture conditions using bone marrow samples from a normal individualand from one patient diagnosed with and treated for acute myelogenousleukemia (AML). Representative cultures were treated with cytokines suchas TNF α alone, IL-4 alone, and TNF α plus IL-4. MPCs from unstimulatedand cytokine-treated cultures were purified using Percoll gradienttechniques disclosed above. Total RNAs were extracted by a standardmethod and analyzed by Northern blotting. This study demonstratedexpression by MPCs of several CAMs, heretofore unsuspected of expressionby BM stroma. These include an embryonic endothelial cell protein Dell(developmental endothelial locus 1), galectin-I, human milk fat globuleprotein (RMFG, lactadherin), and epithelial membrane protein I (EMP 1).Secondly, MPCs from the AML patient expressed significantly lower levelsof mRNA for three CAMs Del-1, galectin-1, and collagen type 1 as well asfor the adipocyte marker adipsin, and to a minor degree themuscle-associated protein caldesmon. On the other hand, mRNA for CAMslike TGF beta-inducible BiGH3, HMFG, osteoblast-specific cadherin 11,and VCAM1 were dramatically increased in AML-associated MPCs. CAMs suchas integrin beta 5, fibronectin, EMP 1 and the muscle-associatedmolecule transgelin are variably increased in diseased MPCs and appearto be unaffected by treatment with cytokines tested. ICAM I wasundetectable at basal level in either patient or normal samples, but wasslightly elevated by TNF α and markedly elevated by TNF α plus IL-4.VCAM1 was mildly up regulated by TNF α alone or IL-4 alone, but markedlyup regulated by TNF α/IL-4 in combination. Also, the MPCs from thepatient were much more sensitive to stimulation by these inflammatorycytokines than were the normal MPCs. These studies establish thatstromal cells in a leukemic patient are functionally defective.

Role of Leptin Receptor in Hematopoiesis Using Human Marrow StromalCells as a Model: The receptor for the product of the obesity gene,leptin, is widely distributed in tissues ranging from central nervoussystem to reproductive system to hematopoietic system. Withinhematopoietic system, OB-R is reportedly expressed on diverse cell typesranging from early CD 34 hematopoietic stem cells to circulatingmonocytes. Leptin acts on monocytes to induce production of TNF α andIL-6, which are powerful regulators of hematopoiesis. However,literature reports on the expression of leptin or its receptor onstromal cells are infrequent. To date, one particular report suggeststhat leptin acts on the stromal cells to enhance their differentiationinto osteoblasts and to inhibit differentiation into adipocytes. Becauseleptin is an adipocyte-generated hormone and because marrow stromalcells represent a unique pluridifferentiated mesenchymal cell typeexpressing some adipocytic features, the expression of the leptinreceptor by these cells was investigated in the hope of revealing itsrole in hematopoiesis.

By Northern blotting marrow stromal cells showed abundant expression ofOB-R, consistent with their adipocytic nature. In terms of regulation,exposure of the stromal cultures to different cytokines revealed aninteresting pattern of OB-R. As shown, G-CSF and TNF α down-regulatedOB-R while IL-4 up-regulated OB-R expression by stromal cells.Simultaneous treatment of stromal cultures with TNF α and IL-4 nearlyabolished OB-R expression. The expression of OB-R was also analyzed atthe protein level by a high-resolution, high capacity 2-D, PAGE system,followed by Western blotting.

More specifically, the method provides the identification of leptinreceptor in human BM stromal cell membrane protein extracts using 2-DWestern blotting. The expression of OB-R was investigated at proteinlevel using 2-D PAGE, followed by Western blotting. Two isoformsdiffering in molecular weight of 2.2 kDa (60.2; 62) and an isoelectricpoint of 0.2 pH unit (5.78; 5.98, respectively) have been identified(the pH was determined by using the values specified by the IPG stripmanufacturer). The ability to subsequently stain the same Western blotwith gold stain allowed precise localization of the immunoreactiveprotein spots of interest on the blot. The gold staining of the blot, byrevealing other protein spots in addition to the immunoreactive spots,has provided the necessary landmarks in turn facilitating subsequentalignment with the silver-stained gel using an appropriate 2-D analysissoftware program (Melanie 3).

This technique has identified two OB-R isoforms that differ in molecularmass by 2.2 kDa (60.2; 62.4) and differ in their isoelectric point by0.2 pH units (5.78; 5.98). The level of macrophage contamination isdetermined by two macrophage markers, CD68 and cathepsin B. The studiesinclude the determination of OB-R expression in a) unfractionatedstromal cells vs. isolated pluri-differentiated mesenchymal progenitorcells; b) unstimulated cultures vs. cultures stimulated with a varietyof cytokine/hormones including leptin itself. The studies also includemass spectrometric characterization of the two OB-R isoforms detected byWestern blotting in order to establish their exact structuraldifferences.

Proteome Analysis of 2-D PAGE Separated Human BM Stromal Cell MembraneProteins: BM stromal cells support the growth and development of normalblood cells as well as providing a haven for malignant leukemia/lymphomacells. Focusing on stromal cell-surface proteins as potentially playinga role in cell-to-cell communication in normal as well as in abnormalhematopoiesis, the mixtures of stromal-cell plasma membrane, and plasmamembrane-associated proteins were analyzed by a high-resolution,high-capacity 2-D gel electrophoresis. The 2-D system described utilizesan immobilized pH gradient gel (pH 4-7) in the first dimension and amini nondenaturing but high-resolution lithium dodecylsulfate-polyacrylamide gel electrophoresis (LDS-PAGE) in the seconddimension. As identified by silver staining, this system has resolvedgreater than 800 protein spots in a pH interval of 2.5 units (4.25-6.75,the isoelectric pH range for most of plasma membrane proteins tomigrate) and a molecular mass range of 10-150 kDa. Equally important,the system is compatible with high sample loads (up to 1.5-2.0 mg oftotal protein in up to 350-μl sample volume). All the protein speciesidentifiable by a silver stain that is compatible with subsequent massspectrometric analysis have been analyzed by a 2-D gel software withrespect to isoelectric point, molecular weight and mass abundance. Thelectin-binding status of these proteins has also been determined bylectin blotting. Lectin blots and Western blots have subsequently beenstained by a gold stain for detection of total proteins on the same PVDFmembrane. Although gold-staining of the Western blot is not as sensitiveas silver-staining of the gel, gold-staining of the Western blotgenerates the necessary landmarks for alignment with the silver stainedgel, facilitating excision of spots of interest from the gel foridentification by MALDI-MS. Representative protein spots were excisedfrom gel and subjected to mass spectrometric profiling (MALDI-MS) and/orsequencing (Nano ESI MS/MS) with subsequent database searching,resulting in a productive identification of ten proteins. The proteindigests are then submitted in a near-ready state for mass spectrometry.Upon receiving the MS data the group performs the database searching.MALDI/MS has been used, which identifies a protein on the basis of itscharacteristic mass sizes, as well as MS/MS studies that provide aminoacid sequences of selected masses to identify proteins with enhancedspecificity and confidence level. This work represents the firstsystematic attempt to analyze BM stromal cell proteins byhigh-resolution 2-D gel electrophoresis and provides the basis for afull-scale proteome mapping of the marrow stromal cells. The presentwork can facilitate the long-term goal of deciphering the hematopoieticsupport functions of BM stromal cells.

Modulation of Stromal Cell Plasma Membrane Protein Expression by TNFα/IL-4: The effects of TNF α/TM on bone marrow stromal cell plasmamembrane protein expression has been tested using the described system.TNF α and IL-4 are regarded as negative regulators of hematopoiesis.Notably these cytokines, especially TNF α, are elevated in marrow plasmaof patients with myelodysplastic syndromes (MDS), the clinical hallmarksof which are anemia, leukopenia and thrombocytopenia (i.e.,pancytopenia). TNF α and IL-4 are thus possible mediators ofhematopoietic dysregulation typical of MDS. TNF α/IL-4 treatment of thestromal cultures induced dramatic changes in the protein profile.Initial studies using plasma membrane protein samples show reducedexpression of at least 7 proteins and enhanced expression of 13proteins.

Analyzing the Insulin-Like Growth Factor System in Human Marrow StromalCells by is 2-D PAGE Analysis of BM Stromal Cell Culture Supernatants:Proliferation and development of normal and leukemic hematopoietic cellswithin bone marrow is regulated by interplay of various classes ofmolecules. These include cell adhesion molecules (CAMs), colonystimulating factors (CSFs), and cytokines as well as growth factorsincluding insulin-like growth factors 1 and 2 (IGF 1 and IGF 2), whichare small peptide homologs of prolinsulin. IGF 1 has knownerythropoietic activity, whereas the function of IGF 2 is less clear.IGF 1 and 2 exert their activities through two types of receptors. Thetype I IGF receptor, a tyrosine kinase receptor highly homologous to theinsulin receptor, binds to IGF 1 and IGF 2 with high affinity. The type11 IGF receptor, a mannose 6-phosphate receptor that lacks intrinsickinase activity, binds IGF 2 with high affinity and IGF I with lowaffinity. The type and number of receptors expressed on a target celldetermine the strength of the IGF signal. One important key tounderstanding the IGFs' role in hematopoiesis is to appreciate howbiological effects of receptors are modulated by larger solubleproteins, the IGF binding proteins (IGFBPs), which share no homologywith the IGF receptors. Because IGFs and IGFBPs play important roles incell growth and proliferation in many tissues, and because marrowstromal cells support hematopoietic growth and development, the patternsof expression of the IGF system components by marrow stromal cellscultured under serum-free conditions is necessary. To this end,unfractionated and purified stromal cells were analyzed, side-by-side,by Northern blotting, under varied stimulatory conditions for expressionof IGFs and IGFBPs with surprising results. IGF 2 is constitutivelyexpressed at a high level by macrophages in Dexter cultures; it is downregulated markedly by TNF ox alone; moderately by TNF α plus IL-4; andunaffected by IL-4 alone. On the other hand, IGF 2 is minimallyexpressed by unstimulated MPCs, but is markedly up regulated by TNF αalone or IL-4 alone; and moderately up regulated by combined TNF α andIL-4. IGFBP4 is abundantly expressed both by macrophages and MPCs and isunaffected by cytokine treatment. In contrast, IGFBPs 5, 7, and 10,selectively expressed by MPCs, show no evidence of expression bymacrophages and are unaffected by cytokine treatments. IGF 1 and theprecursor to IOFBP 3 are not expressed in either macrophages or MPCs,either constitutively or after stimulation with TNF α, IL-4 or both. Ininitial studies, bone marrow mononuclear cells expressed none of theIGFs or IGFBPs tested. These results provide important insights into theoperation of the IGF system in stromal cells and it is likely thatpotentially novel IGFBPs can be uncovered by ligand blotting studies.

The present invention provides a large-format 2-D gel electrophoreticsystem for reproducible separation of MPG proteins and to prepare 2-DPAGE protein maps for normal bone marrow-derived MPCs (untreated andtreated with representative cytokines, e.g. TNF α or IL-4) and for MPCsderived from patients with representative pre-leukemic and leukemicconditions. The pre-leukemic conditions include myelodysplasticsyndromes (MDS) and the leukemic conditions include chronic myeloidleukemia (CML), acute myeloid leukemia (AML), chronic lymphocyticleukemia (CLL), acute lymphocytic leukemia (ALL), and multiple myeloma(MM). The protein samples can consist of culture supernatants/secretedproteins; extracellular matrix (ECM) proteins; plasma membrane proteinssolubilized using a three-step differential extraction protocolemploying conditions of progressively increasing solubility; and wholecell lysate proteins similarly solubilized using the three-stepdifferential extraction protocol. This subproteome approach not onlysimplifies the 2-D PAGE electrophoretic protein patterns but alsoreveals additional proteins, which would otherwise have gone undetected.

Molecular Analysis Assay Involving the High-Resolution 2-D PAGE and MassSpectrometric Identification of Gel-Separated Proteins: The completionof human genome project has provided a huge proteome database includingthe theoretical mass databases generated on the basis of site-specificcleavage employing proteolytic enzymes, such as trypsin and others. Theavailability of highly sensitive biological mass spectrometers togetherwith the capability of bioinformatics to search extremely large amountsof data and identify the relevant proteins matching the massspectrometry data provides the basis for the current excitement inproteomics. The focus of the interest is the BM MPC proteome asexpressed under varied functional and disease states. The goal of thepresent invention is to identify BM MPC proteins that have possiblefunctional and/or pathologic significance, that is, those proteins thatshow altered levels of expression in response to cytokine treatments andvarious leukemic states.

Until recently, the focus of the laboratory has centered on isolationand characterization of BM stromal cell adhesion molecules using a novel2-D cell blotting technique. For this purpose, applicants haveestablished an analytical 2-D mini gel system that separates stromalcell plasma membrane proteins using 18-cm long 4-7 pH range IPG stripsin the 1^(St) D (Amersham Pharmacia Biotech). Subsequent to IEF, the IPGstrip is cut into appropriately small pieces and subjected to 2 Dseparation using nondenaturing lithium dodecyl sulfate-polyacrylamidegel electrophoresis (LDSPAGE) and mini gels. The reason for using minigels in the 2^(nd) D is that they are compatible with a downstreamfunctional assay involving cell adhesion. The stromal cell membraneproteins are blotted on to a PVDF membrane and assayed for hematopoieticcell-binding proteins directly on the blotting membrane. The system canbe extended using 17-cm long 3-10 pH range IPG strips (Bio-Rad) forseparation of stromal cell culture supernatants, ECM proteins, and wholecell lysates. As detected by silver staining of the gels, and analyzedby appropriate software (GelLab II or Melanie 3) this 2-D system hasresolved greater than 800 membrane protein spots within a pH interval of2.5 units (4.25-6.75) and a MW range of 10-150 kDa. Similarly, the ECMsamples showed 475 spots; and conditioned media from BM stromal cellcultures grown under serum free conditions showed 524 spots. Notsurprisingly, the total cell lysate of BM stromal cultures showed only553 spots, most likely representing the abundant housekeeping proteinsand masking detection of many functionally relevant proteins. Theseobservations provide the rationale for the proposed subproteome approachinvolving the use of differential solubilization of sample proteins andmultiple large gels. Membrane proteins thus far identified by massspectrometry followed by database searching; proteins are identified bystandard Western blotting. Select IGF binding proteins were identifiedby ligand Western blotting. The blotting shows the identification ofIGF-binding proteins (IGFBPs) using 2-D ligand blotting. The conditionedmedia from BM stromal cultures grown under serum-free conditions wereconcentrated using Microcon concentrator, and proteins were fractionatedusing a high-resolution 2-D PAGE. The separated proteins wereelectroblotted onto PVDF membrane and subjected to Westernligand-blotting assays using 1-125 labeled IGF-2, resulting in theidentification of a series of IGFBPs (up to 30 spots). Notably, TNF αtreatment of the cultures down-regulated two LGFBPs and up-regulatedIGFBP labeled 6.

By necessity the protein work began on BM stromal cells using anondenaturing (LDS-PAGE) mini gel system that contained noreducing/alkylating agents. To preserve the function of 2-D gelseparated proteins many otherwise powerful sample preparationmethodologies designed for proteomic studies (such as multiplesurfactant solution, MSS) were avoided. While mini gels are convenientand allow comparison and information transfer to large-format gels, theyare less sensitive.

Subproteomes According to Sample Prefractionation: In order to be ableto identify the low-abundance proteins implicated in the regulatory andpathologic processes, a number of approaches to prefractionation of thewhole cell lysates have been described. Applicant studied thesubproteomes of secreted proteins from BM stromal cell culturesupernatants as well as ECM proteins. Notably, the ECM protein samplescan be a rich source of functionally relevant cytokines and chemokinessince the latter are known to mediate function by binding to ECM. Inaddition, the plasma membrane and whole cell samples were subjected tothe 3-step sequential solubilization protocol shown. The solubilizingsolutions can be prepared in-house or purchased commercially (Bio-Rad).The first step involves the use of Tris base, which can solubilize theperipheral membrane proteins and cytosolic proteins. These proteins arelyophilized and subsequently solubilized prior to 2-D PAGE in a standardsolubilizing medium (the modified O'Farrell cell lysis solutioncontaining urea, CHAPS, DII, Iris, ampholytes and appropriate proteaseinhibitors). The resulting pellet can also be solubilized in thestandard 2-D solubilizing medium and subjected to 2-D PAGE. Because thestandard solution cannot solubilize some proteins, the membrane-richpellet is finally solubilized in a potent multiple surfactant solution(MSS) consisting of urea, thitheea, Cl-LAPS, zwittergent 3-10 andtributyl phosphine (TBP) in addition to Iris base and ampholytes that iscompatible with subsequent IEF. The MSS has been shown to solubilize thehydrophobic proteins with as many as 12 transmembrane regions (TMRs),facilitating their 2-D analysis. Another final step incorporating 1% SDSin boiling sample buffer can be used to test by 1-D SDS-PAGE if anyproteins remained unsolubilized after these extractions (notably, SDSextract is unsuitable for 2-D PAGE analysis since SDS interferes withIEF). The prefractionation step clearly reduces the complexity of thesample. Thus, the serial extractions not only simplify the gel imagesand reduce spot overlapping frequently encountered in single-stepextractions but also correlate closely with the cellular location ofspecific proteins, providing clues to their function. Theprefractionation strategy can be extended to enriching low-abundanceproteins in culture supernatants by selective removal of contaminatingalbumin using an Albumin Depletion Kit (containing Cibachron Blueresins) (Genomic Solutions, mc). Similarly, membrane glycoproteins canbe enriched by a Glycoprotein Enrichment Kit (containing lectins) priorto 2-D PAGE analysis (Genomic Solutions, Inc.).

Subproteomes According to Overlapping pH Gradients: Using a series ofmedium-range and partially overlapping pH gradients (3-6, 5-8, 7-10,each 17-cm long) (Bio-Rad) can enhance reproducibility and resolution bycreating “virtual” gels with up to 40 cm equivalent of PI separationacross a pH 3-10 range. Alternatively, a combination of two pHgradients, pH 4-7 and pH 6-11, each 24 cm-long (Amersham PharmaciaBiotech) can be used, also providing a “virtual” separation distance of40 cm across a pH range of 4-11. These strips accept micropreparativesample loads (1-2 mg). Notably, a given sample of cells yields a totalof 8 protein samples. These samples include one protein sample composedof conditioned medium, one protein sample comprising of ECM proteins,three protein samples derived from plasma membrane lysates and threeprotein samples derived from total cell lysates, following applicationof a three-step protein extraction protocol to purified plasma membranesand total cells. Eight protein samples can thus translate into 24 largeformat (18 cm) gels corresponding to three overlapping 1st D gels; or 16extra large format gels (24 cm) corresponding to two overlapping I st Dgels. Proteomics is no longer considered a single 2-D gel study. Takingadvantage of the common spots in the 2nd D corresponding to overlappingregions, PDQUEST software can allow “stitching together” of theconstituent gels, creating the so-called “cyber gel” providing acomposite map for each protein sample. The data generated can be storedin an internet-accessible 2-D PAGE database in the form of 8 submaps.Three of these submaps correspond to plasma membrane proteinsrepresenting 3-step solubilization; one of them corresponds to secretedproteins; one of them corresponds to ECM proteins; and 3 of themcorrespond to total cell lysate proteins representing 3-stepsolubilization. These submaps can be linked to a master synthetic gel, aconglomerate of the submaps, representing the so-called “cyberproteome”of MPCs. Given the ability to run up to 12 IPG strips per 1st D gel(using IPGPhor) and 10 to 12 large or extra large SDS-PAGE gels per2^(nd) D gel run (using Hoefer DALI and Ettan DALI II, respectively),the resulting number of gels can be well within the manageable workloadof one person (36). Although not easily accessible now, some innovativetechnological developments are on the horizon, e.g., development offluorescence 2-D difference gel electrophoresis (DIGE), which couldminimize the tedium. Unlike the current practice of running differentprotein samples on separate gels, and then staining and comparing thegels, DIGE technology uses matched, spectrally resolvable dyes (e.g.,Cy2, Cy3 and Cy5) to label protein samples prior to 2-D separation.Differentially labeled protein samples are mixed and co-separated by 2-Delectrophoresis, allowing analysis of at least three samples on a singlegel. Gels are scanned and proteins are subjected to image analysis usingappropriate software. Alternatively, one can use a highly sensitivesilver stain to visualize the proteins after electrophoretic separation.Notably, the silver stain is compatible with subsequent massspectrometry analysis.

Follow-Up Strategy for 2-D PAGE Using the So-Called Ultrazoom LPG Gelswith Narrow-Range pH Gradients: Commercially available narrow-range IPGstrips include pH 3.54.5; 4.0-5.0; 4.5-5.5; 5.0-6.0; 5.5-6.7. These areavailable as 18 cm and 24 cm-long strips, consequently allowing spanningof 1 pH unit over a distance of 18-24 cm and providing extraordinaryresolution. By using narrow pH gradients (IpH unit) up to 10 mg ofprotein would be loaded onto a single IPG gel strip, either by repeatedsample cup application or by in-gel rehydration without incurringvertical or horizontal streaking. Employing a combination of suchnarrow-range overlapping IPG strips, one study utilized up to 40 2-Dgels for analysis of a single protein sample. The preference is not tofollow such extreme approach but rather to use these gradients as abackup in situations where a functionally relevant protein is firstdetected by the front-end strategy but could not be studied by massspectrometry for lack of adequate resolution or due to overlappingspots. The 24-cm long narrow IPG strips can be subjected to 2^(nd) Dusing correspondingly extra large slab gels (the required precast,plastic-backed gels can be purchased from Amersham Pharmacia Biotech).However, the “giant 2-DE” 30×40 cm size gels are impractical to handle.The situations for the use of narrow range pH gradients includesituations like detection of proteins by Western blotting usinganti-phosphotyrosine antibodies or Western ligand blotting using labeledIGF 1 or 2, which are probably more sensitive than silver staining.Consequently, these assays identify the functionally relevant proteinsbut without providing the actual identity of the individual proteins.Because the front-end strategy can at least provide the range of thephosphoprotein or the IGF-binding protein identified, on the basis ofthis information samples can be subjected 2-D PAGE using the appropriateultrazoom IPG strip, which as indicated above can permit loading ofseveral mg of protein sample. Extra large precast slab gels (26×20 cm)with plastic backing suitable for running the 24-cm long ultrazoom IPGstrips and the appropriate electrophoretic system (Ettan DALI II 2dimension electrophoresis system) that runs up to 12 of these gels arecommercially available (Amersham Pharmacia Biotech).

Summary of Subproteome Strategy: The subproteome approach involves: 1)Cellular fractionation involving isolation of purified MPCs. 2)Subcellular fractionation involving preparation of functionally relevantprotein sets. These include: 2a) secreted proteins such as colonystimulating factors (CSFs), cytokines, etc in the conditioned media; 2b)ECM proteins such as cell adhesion molecules (CAMs), etc; 2c) plasmamembrane proteins such as various receptor molecules, CAMs andcomponents of cell signaling systems, etc; 2d) finally, whole celllysate proteins that include some of these proteins plus cytosolic andnuclear proteins. The cytosolic and nuclear proteins can be a richsource of target proteins for phosphorylation with a regulatoryfunction.

2-D PAGE Data Capture and Analysis: 2-D PAGE data capture and analysiscan be performed using standard equipment and protocols. Silver-stainedgels can be scanned using an imaging densitometer and processed withQuantOne software (Bio-Rad) whereas gels stained with fluorescent SyproRuby (with 450 nm in the excitation range) can be scanned using a STORM860 gel and blot imaging system and processed with ImageQuant Solutionssoftware (Amersham Pharmacia Biotech). A number of factors, includingdifferences in sample preparation and loading, staining and imageacquisition can influence the reproducibility of 2-D gel proteinseparation. Quantitative data are reported as spot volumes (integratedspot densities). In experiments comparing replicate 2-DE patterns of thesame sample or 2-DE patterns of samples from different individuals, thespot volumes in each pattern are scaled to correct for differences inthe total amount of protein loaded onto each gel. These variations arecompensated by accurately comparing the quantity of any spot acrossmultiple gels. These operations can be performed using a dedicated 2-Dgel analysis software, Melanie 3. This program can analyze suchvariations by scatter analysis and can compensate for varying stainingabsorption across proteins by normalizing protein expression change.Varying stain intensities and sample sizes can be compensated for byrelative spots quantification. The Melanie 3 software also has thecapability to merge several gel electrophoretic patterns from the samesample into a composite gel, providing fine control over the includedproteins. Finally, the software can compensate for gel distortionscaused by variations in protein migration through alignment of the gels.

Mass Spectrometric Instrumentation: Examples of such mass spectrometersinclude, but are not limited to, Voyager DE Pro (Applied Biosystems,formerly Perceptive Biosystems, Inc.) and QSTAR (Applied Biosystems).Voyager DE Pro is a matrix-assisted laser desorption time-of-flight massspectrometer (MALDI-IOF) that can be operated in a linear mode for theanalysis of large biomolecules or in a reflector mode forhigh-resolution analysis of smaller molecules, i.e., peptides. TheMALDI-TOF instrument also utilizes delayed extraction technology thatresults in greatly increased resolution, sensitivity and mass accuracy.This is the instrument of choice for high throughput analysis, with acapacity of up to 100 samples per sample plate. On the other hand, theQSTAR is a hybrid quadrupole-quadrupole-time-of-flight massespectrometer. Samples are introduced in solution and are ionized byelectrospray. For samples requiring the highest sensitivity, Dr. Jacksonutilizes a low flow rate (25 nl miff) electrospray callednanoelectrospray, typically requiring only 1-2 μl of a solution forsample analysis. The QSTAR instrument yields data quite similar to thoseobtained from the MALDI-TOF instrument, except that the QSTAR dataanalysis is somewhat more complex due to the multiple charging ofpeptides by the electrospray process compared to the single chargingapplied to peptides on the MALDI-TOF instrument. One importantadditional characteristic of the QSTAR is its ability to determinestructural information from sample molecules by tandem MS/MS. This isachieved by effectively “purifying” selected molecules within the massspectrometer's first quadrupole section. For analysis of peptidesproduced by tryptic digestion, a single MS experiment is initiallyperformed to determine the masses of components present in the mixture.Next, MS/MS experiments are carried out to select specific peptides forde novo amino acid sequence determination. Typically 2 μl of peptidemixture is sufficient for determining the sequences of ten to twelvepeptides.

Mass Spectrometric Analysis by MALDI: The scheme for mass spectrometralanalysis of in-gel tryptic digests of proteins for the purpose ofprotein identification consists of several steps. First, the peptidesextracted from the gel must be cleaned and concentrated. The cleanup isnecessary to remove residual detergent and other non-peptide materialsthat can interfere with the analysis of the tryptic peptides. This stepinvolves binding of the peptides to a Microcon-SCX adsorptivemicroconcentrator. This is a cation exchange membrane held within amicrocentrifuge device. At low pH, the peptides bind to the negativelycharged membrane, while uncharged or negatively charged molecules passthrough. After a brief wash, the peptides are eluted from the membranein two 25-μl steps of 1.5 N ammonium hydroxide in 1:1 methanol/water.The samples are then speed-vac dried for 10 minutes, and fresh solventis added for additional treatment to concentrate the sample prior to MSanalysis. Initially, all samples can be analyzed by MALDI-TOF MS. Forthis analysis, the sample from the Microcon-SCX elution can be dissolvedin 0.1% trifluoroacetic acid (TFA) in water and loaded on a ZipTipC 18Pipette tip. The tip is then washed with the same solution and thepeptides are then eluted directly onto the MALDI-TOF sample plate with 2μl matrix solution (cyano-4-hydroxy-cinnamic acid, 10 mg/ml in 0.1% TFAin 1:1 acetonitrile/water). The spotted sample is dried at roomtemperature for at least five minutes before the sample plate is loadedin the instrument. The instrument calibration is performed externally bythe addition of a calibration mixture to the sample plate. Samples arecalibrated internally if the known tryptic autodigestion peptides areobserved in the sample. This can be used as long as the specific type oftrypsin used in the proteolytic digestion step is known. After datacollection, the data can be further processed in two ways. First, thedata can be treated by noise reduction software and second, it can bedeisotoped. Software for both operations of these programs are standardfeatures of the Data Explorer system provided with the Voyager DE Promass spectrometer. The obtained peptide mass data can be subjected topeptide fingerprint analysis utilizing one of the protein databasesearch sites on the Internet, such as Mascot or MS-Fit. While each ofthese search sites has access to several databases, one can initiallyselect either OWL or NCBlnr. One can search the database with a standardset of criteria without using a species filter. The practice is toselect three variable modifications to allow for conversion of peptideN-terminal glutamine to pyroglutamate, and oxidation of methionineresidues; allowing for up to one missed cleavage. Neither the protein MWnor the PI can be used as a search parameter (these, however, can beused for subsequent validation of the matched protein). Also importantis that expected peptide masses of known potential “contaminants” suchas keratin and trypsin can be excluded from analysis. Finally, thepeptide mass tolerance can be set to +1-0.15 Da relative to themonoisotopic MW of the singly charged peptide ion. Positive databasehits are scored with a MOWSE number. The higher the number of hits thegreater the confidence level. The database search algorithm relates thesignificance level for a given search. If a high MOWSE score is obtainedindicating an unambiguous match, one can consider the protein positivelyidentified, otherwise the sample can be subjected to analysis by use ofthe QSTAR mass spectrometer.

Mass Spectrometric Analysis by Nano ESI MS/MS: Samples that requireanalysis utilizing the QSTAR, following cleanup by Microcon-SCXadsorptive microconcentrator, can be concentrated by binding the peptidemix to a small amount of POROS R12 reversed-phase C18 chromatographicsupport packed into a nanopurification capillary. The packed capillarycolumn volume is 10-15 nl. The sample, dissolved in 10-p.l of 5% aceticacid in water, is applied to the capillary by use of a ten-μl gelloading pipette tip. A brief centrifugation forces the liquid down thecapillary so that the peptides can bind to the support. The support isthen washed with 10-15 p.l of 0.5% acetic acid in 1:50 methanol/water.The peptides are eluted from the purification capillary into a nanospraycapillary by the addition of 2 μl of 0.5% act id in 1:1 methanol/waterfollowed by brief centrifugation with the nanospray capillary stackedjust below the purification capillary in a micropurification holder (MDSProtana). Initially, data for a single MS run is collected. The peakmasses are labeled and peptides are selected for potential MS/MSsequencing by locating those that appear to be doubly charged. Mostpeptides resulting from a tryptic digest can have a significant doublycharged form, which is ideal for MS/MS sequencing. The first quadrupoleof the QSTAR is tuned to pass a 2 Dalton window for the pre-selecteddoubly charged peptide ions, one at a time, for fragmentation bycollision with low-pressure argon gas in the second quadrupole.Collision energy is adjusted for each peptide to obtain the bestpossible MS/MS spectra. Data are collected long enough to get goodquality spectra. After MS/MS spectra are collected for all selectedpeptides, the data are manually interpreted. Internet protein databasesearches are performed similar to that for MALDI-TOF peptidefingerprint, except that the search is a partial amino acid sequencesearch with mass information (i.e., Mascot, employing Sequence Queryformat). The search criteria cannot screen for a species or a protein MWor PI (which, however, can be used for subsequent validation of theprotein matched). Also important is that expected peptide masses ofknown potential “contaminants” such as keratin and trypsin can beexcluded from MS/MS analysis. One missed cleavage can be allowed and twovariable modifications can be selected, carbamidomethylation of cysteineand oxidation of methionine. The tolerance of the peptide monoisotopicmass can be set to +1-0.3 and the MS/MS tolerance can be set at +1-0.2.This type of search generally requires only two or three peptidesequences consisting of three of the amino acids per peptide to obtain astatistically significant match (a high MOWSE score). Once a match isidentified, a list of the matched peptide's theoretical MS/MS fragmentscan be generated to compare with the observed fragments to furtherconfirm the correctness of the match.

Establishment of Large-Format 2-D PAGE Protein Maps for MPCs Derivedfrom Normal BM: The normal cell samples include, A)-Untreated normalMPCs; B) Normal MPCs treated with TNF α; C) Normal MPCs treated withTNF- and IL-4. Each cell sample can generate a total of 8 proteinsamples, 1) culture supernatants/secreted proteins (1 protein sample);2) extracellular matrix (ECM) proteins (1 protein sample); 3) plasmamembrane proteins solubilized using a three-step differential extractionprotocol employing conditions of progressively increasing solubility (3protein samples); 4) whole cell lysate proteins similarly solubilizedusing a 3-step differential extraction protocol (3 protein samples).Each protein sample can generate 3 large format 2 D gels (correspondingto 3 medium-range, overlapping IPG gradient gels, pH 3-6; 5-8; 7-10).This means each cell sample can generate 24 large format 2 D gels,leading to generation of at least 72 large format gels for analysis ofnormal MPCs. To account for duplicate or triplicate samples, the gelnumber falls in the range of 200-300.

Establishment of Large-Format 2-D PAGE Protein Maps for MPCs Derivedfrom BM Involved with Representative Pre-Leukemic and LeukemicConditions: The disease-associated MPCs include those from MDS, CML,AML, CLL, ALL, and MM. As above, each MPC sample can generate 24large-format 2 D gels. With 6 such diseases being studied, the gelnumber can reach 144. To account for duplicate or triplicate samples,the gel number falls within the range of 400-500. The use of IPGPhor,together with ready-made IPG strips, permits sample in-gel re-hydrationand performance of unattended IEF overnight by adding automation to the2-D procedure.

Using High-Resolution Proteomics and with the Added Power ofHigh-Throughput Robotics, Identify on a Larger (Semi-Comprehensive)Scale the MPC Proteins that are Differentially Expressed in Conditionsthat Simulate Pre-Leukemic Bone Marrow (Following Stimulation withDifferent Cytokines), and in Actual Pre-Leukemic Disorders (MDS) as wellas in Overt Leukemias (CML, AML, CLL, ALL, MM): A robotically guidedsystem facilitates excision of protein spots (by a spot cutter orpicker) from 2-D PAGE gels, transfer of protein samples to 96-wellmicroplates, and automated protein digestion in the microwells. Such asystem reduces the time and labor relative to manual procedures andprovides high throughput while minimizing keratin contamination fromhuman skin, a frequent problem in proteomics research. The preferredmethod is to excise all spots from a gel but to process only the spotsof interest, storing the remaining excised proteins frozen at −70° C.for a later use. The robotic components can include MALDI slide spotterin addition to an automated protein spot picker and digestion station.

Example 8

Methods: The present study involved microarray analysis of 23 samplesand a corresponding number of chips. The samples were obtained from 4normal healthy adult human subjects, consisting of mixtures ofunfractionated stromal cells (collective USCs or cUSCs, 8 samples),Percoll gradient-purified MPCs (collective MPCs or cMPCs, 5 samples) andsingle-cell MPCs (sMPCs, 10 samples) obtained by laser-capturemicrodissection (LCM). The study design allowed for adequate controlsand replicates appropriate for a comprehensive gene expression profilingof normal BM stromal cells. The isolated single stromal cells wereselected on the basis of morphology. Wright-Giemsa stained cytospinpreparation revealed characteristically large cells with a relativelyirregular nucleus and cytoplasm compartmentalized into ectoplasm andendoplasm. Subsequently, applicant identified a hematoxylin stain as asubstitute for Wright-Giemsa stain. The hematoxylin stain is simpler touse and provides morphologic detail sufficient to allow recognition andisolation of these cells by laser capture microdissection and does notinterfere with the downstream microarray testing (see details underMaterials & Methods). The photomicrographs of 10 stromal cells that havebeen subjected to microarray testing are shown in FIG. 15. To serve ascontrols and facilitate comparison, applicant analyzed side-by-side 8samples of unfractionated stromal cells that are “contaminated” by up to35% macrophages and 5% hematopoietic cells (referred to collective USC,or cUSC), and 5 samples of Percoll-gradient purified stromal cells, upto 95% pure (referred to collective MPC, or cMPC to distinguish fromSMPC). RNA isolated from sMPC samples was subjected to 2 rounds ofamplification using RiboAmp kit (Arcturus, Inc) prior to in vitrotranscription (IVT). In contrast, RNA samples isolated from cUSCs andcMPCs were used without amplification for IVT. The subsequent steps ofmicroarray testing were standard for all 3 types of samples and areschematized as follows: Preparation of total RNA→generation ofcDNA→preparation of ds cDNA→in vitro transcription intocRNA→fragmentation of cRNA→hybridization of target RNA to a microarrayof known genes (Affymetrix U95Av2 oligonucleotide microarray, with12,625 probe sets)→Signal quantification and first-tier analysis usingthe microarray quantification software, Microarray Suite (MAS v. 5,Affymetrix, Inc). The presence of a gene within a given a sample wasdetermined at a detection p-value of <0.05, according to the statisticalexpression analysis algorithm employed by MAS v.5, and was graded absent(A), marginal (M) or present/positive (P).

Assessment of RNA Amplification Method: Since single-cell microarraysare relatively novel, applicant critically reviewed the data withrespect to two important statistics that would reflect on thereliability of RNA amplification assay procedure employed beforeanalysis involving data-mining techniques. a) 3′:5′ ratios ofhousekeeping control genes: As shown in Table 9, these ratios were closeto 1 in the standard unamplified samples, whereas, they were increasedin the amplified samples. Although this is to be expected due topreferential amplification toward 3′ end, since amplification may notproceed all the way up to 5′ end, applicant wanted to exclude possiblesample degradation. For reasons unclear, in the case of ACTB (betaactin), the 3′:5′ ratios were highly variable across single cell MPGsamples. In any event, the 3′:5′ ratios in case of GAPD (glyceraldehyde3-phosphate dehydrogenase) were relatively tight, suggesting no evidenceof sample degradation. Furthermore, both GAPD and ACTB gene probes thatwere employed as part of the standard gene probe set yielded relativelystable signals across replicates in each sample type, which is furtherevidence of intactness of RNA samples targeted for microarray analysis.b) Number of genes present or detected: As outlined in Table 9, theamplified single cell MPG RNA samples expectedly showed significantlylower number of genes compared to the standard RNA samples (on average34% vs. 46% of the genes etched on the array). The fact that the numberis relatively constant across single cell replicate samples is furtherindication of the reliability of the data. Notwithstanding theshortcomings of the amplification procedure, it is important to bear inmind that the conclusions are based only on those stromal cell genesthat are detected commonly across unamplified cMPCs and cUSCs as well asin amplified sMPCs (but do not include the genes undetected or the genesselectively detected in sMPCs).

Data Mining and Reproducibility of Overall Procedures: The thrust of thepresent invention is to identify genes that are relatively uniformlyexpressed across normal untreated bone marrow stromal cell samples,regardless whether they are of single cell type or collective cellsamples, purified or unpurified. As detailed under Materials & Methods,GENESPRING was used to achieve the following data-analysis objectives:a) Filtering for genes reliably detected in each sample group byeliminating the genes with weak expressions that are statistically closeto the background estimate. b) Filtering for genes that are active or“present” across replicates in each sample group. c) Exclusion of geneswith weak expressions from genes “present” in each sample group. d)Preparation of master stromal cell gene list by intersecting gene listsfrom step (c) (as shown in FIG. 16). These steps have led toidentification of a list of 2755 genes that are detected in at least 7of 8 cUSC samples AND 4 of 5 cMPC samples AND 9 of 10 sMPC samples,i.e., in at least 20 of 23 stromal cell samples investigated. The mainconclusions of the present report are based on this “stromal cell genelist” that is broadly representative of all 3 types of stromal cellsamples investigated, and not on a gene list that is selective to sMPCs.A hallmark of the quality of microarray data can be discerned fromhierarchical cluster analysis of replicates, which involves theprinciples of vector algebra. An array of numbers representingexpression levels of a particular gene in terms of normalized signalintensity in a group of samples is considered a gene expression vector.Likewise, an array of numbers showing expression levels of a group ofgenes by a particular tissue sample is considered a tissue expressionvector. In the case, applicants have 2755 gene expression vectors and 23tissue or sample expression vectors. These vectors are amenable toalgebraic treatment, facilitating calculation of similarity between anytwo gene- or tissue-expression vectors on the basis of a correlativemetric or a similarity-measure employed, e.g., Euclidean angle. Groupingtogether of two samples on the basis of these principles signifies thatthey are most closely related out of all the samples in terms of theiroverall pattern of gene expression. Construction of a bone marrowstromal cell hierarchical tree has enabled visualization of global geneexpression patterns across replicates and conditions. As shown in FIG.17, stromal cell genes that are expressed at a relatively lower level inamplified samples (sMPCs) are clustered to the left of gene tree, genesthat are more strongly expressed in sMPCs are prominently figured in themiddle of gene tree, and genes that are expressed approximately at samelevel as in unamplified samples (cMPCs and cUSCs) are clustered to rightof gene tree. Even more important as noticeable on the sample orexperiment tree, hierarchical clustering segregated the members of eachsample type into a separate group (cMPC, cUSC and SMPC). Note withineach sample type, corresponding subject replicates clustered togetherwith minor exceptions. This is a reflection on the reproducibility ofthe overall assay-procedures employed, which encompass a variety ofstages and steps in addition to target RNA amplification prior to invitro transcription (see Materials & Methods for details).

Finally, it is important to keep in mind that the measured level of atranscript following amplification does not necessarily provide aquantitative estimate of gene expression, but only provides aqualitative indication that the gene is transcriptionally active, whichby itself is sufficient grounds for the conclusions arrived at in thepresent report. As shown in FIG. 18, the expression of genes within thestromal cell gene list ranges from 0.2 to 6 (on the log scale) inunamplified samples (cMPC and cUSC) and from 0.02 to 36 in amplifiedsamples (sMPC), thus showing much greater variability in the amplifiedsamples. For illustration purpose, the coloring of gene expressioncurves (following the linear color bar shown on the right) was based onthe gene expression pattern of a particular single-cell sample, SCA1.Note that the genes that are detected at a low level in this sample (asindicated in blue) are not necessarily expressed at a low level inunamplified samples (as read by the log scale on Y-axis). In fact, asignificant number of them are expressed at a high level in theunamplified samples. This finding together with the observation thatamplified samples detected about 34% of genes as opposed to unamplifiedsamples detecting about 46% of genes tested (Table 9), strikes acautious note that some genes do not amplify at all by the method used,and other genes amplify to a sufficient degree to be detectable (shownin blue), while some other genes amplify to a degree equal to (inyellow) or surpassing (in red) the amounts in the collective samples.(The curve shown in white is the housekeeping gene, GAPD.) Thestatistical algorithm as implemented in the latest version of MicroarrayAnalysis Suite (MAS v.5) determined that a gene within a given sample aspositive, regardless of grading. To overcome the limitations of theamplification procedure employed, applicant focused only on genes thatwere positive in at least 20 of the 23 stromal cell samplesinvestigated.

A stromal cell gene list is expected to be representative of typicalstromal cell gene expression profile. Such master gene list forms thebasis for derivation of all other stromal cell gene lists, organized inaccordance with lineage or functional categories. As depicted in FIGS.19A-19F, and listed in Tables 10 through Table 17, that were preparedaccording to lineage/functional assignment, the findings show thatisolated single cells simultaneously express genes associated withdiverse mesenchymal cell lineages, namely osteoblasts, muscle cells,fibroblasts, adipocytes, epithelial cells, endothelial cells, nervecells and glial cells, providing confirmation of the existence of apluridifferentiated progenitor cell type. By definition the stromal cellgenes are present in at least 4 of 5 collective MPC samples AND 7 of 8collective USC samples AND 9 of 10 single cell MPC samples;consequently, they are active in at least 20 of 23 samples tested,representing a typical genomic profile of stromal cells. The followinggene lists are sub-lists of the master stromal cell gene list consistingof 2,755 genes. The stromal cell gene list contains a number of genesthat are capable of causing endothelial differentiation andvasculogenesis within the marrow microenvironment; however, these genesthemselves are not necessarily endothelial cell markers. In fact,stromal cells express a gene, EDF1, the expression of which inverselycorrelates with endothelial cell differentiation within the stromalcells. Of the 67AFFX control genes present on the U95A v2 chip, 22 genesare detected in at least 7 of 8 cUSC samples, 24 genes are detected in 4of 5 cMPC samples and 19 genes are detected in at least in 9 of 10 sMPCsamples. Thirteen of these genes are present in the stromal cell genelist, i.e. in 20 of the 23 samples investigated.

As evident from these gene lists, note that an isolated single stromalcell simultaneously expresses transcripts for epithelial andneuroectodermal cell types as well. Departing even further from theinitial thinking, the findings add to the evidence that the MPCs withinthe Dexter system might represent a form or stage of the progenitor cellthat is common to nonhematopoietic and hematopoietic cells. As depictedin FIGS. 20A-20F, and listed in Table 18 through Table 21, the isolatedsingle stromal cells express transcripts that are typical ofhematopoietic cells, in particular precursor B cells. That BM stromalcells express CD10 (CALLA) is not novel since BM stromal cells as wellas endometrial stromal cells and normal breast myoepithelial cells areknown to express CD10. However, the expression of CD19, CD79A andimmunoglobulin enhancer binding factors E12/E47 (proto-oncogene TCF3) byBM stromal cells is unforeseen, and forms the basis for postulating theexistence of a common progenitor with B cell lineage. B-cell progenitorstypically display the phenotype, (CD45±, CD34±, CD20±, (CD10+, CD19+,CD79A+, HLA-Dr+), which as reported here is also displayed by isolatedsingle stromal cells at least at the transcriptome level.

CD45 positivity by cMPC and cUSC samples is attributable to coexistingor contaminating hematopoietic cells in these samples as evidenced byconcurrent positivity for myelomonocytic markers CD13, CD33 and CD14(Table 21). However, a products or transcripts for CD45 and CD19 aremost likely present in stromal cells at a basal level that is beyond thedetection limits of conventional techniques, e.g., immunocytochemistryand Northern blotting, respectively. Conceivably, two rounds ofamplification prior to IVT sufficiently increased their transcriptlevels to be detected by microarray analysis. In fact, the CD45 levelswere several-fold lower in cMPC and cUSC compared to CD45 levels insMPC, and CD19 was undetectable in unamplified samples. Finally, notethat CD45 and CD19 are not isolated examples in this regard sinceapplicant has identified at least 200 other genes that are uniquelypresent across sMPC samples but undetectable in cMPC and cUSC (FIG. 16).

The Issue of Stromal Cell-B Cell Connection:

Although no analog of Bursa of Fabricius exists in mammals, bone marrowis generally regarded as the site of B-cell generation. While the Dextertype stromal cell culture system was devised for investigation ofhematopoiesis, specifically myelopoiesis (see Introduction), Whitlockand Witte developed another system for the study of B-lymphopoiesis.Whitlock-Witte cultures, like Friedenstein cultures, are grown in theabsence of hydrocortisone and horse serum. When stromal cell layers inW-W cultures are seeded with fresh bone marrow as a source of B cellprecursors or with purified B cell precursors, the latter thendifferentiate into mature B cells. On the other hand, although Dextercultures do not promote B cell maturation, Dexter cultures do contain Bcell precursors, which upon switching of culture conditions from thoseof Dexter to Whitlock-Witte, differentiate into mature B cells. While BMstromal cells in one form or another are definitely known to supportB-lymphopoiesis, it has not been so clear as to whether stromal cellsactually give rise to B-cell precursors. Evidence for a progenitor cellcommon to stromal cells and hematopoietic cells has been coming to lightpiecemeal in the form of isolated reports. 1) Singer J W et al in 1984in the course of investigating bone marrow cultures from human patientswith clonal myeloproliferative disorders showed that thenonhematopoietic stromal cells were derived from the same clonalprogenitors that were involved by the hematopoietic neoplasm, asrevealed by G6PD marker analysis. 2) Huss R et al in 1995 in the courseof investigating a canine BM stromal cell line showed that the adherentstromal cells had “turned” into nonadherent hematopoietic cells,especially when the latter were cultured in presence of stem cellfactor. 3) Pessina et al in 1997 showed that a particular murine stromalcell line upon stimulation with bFGF, expressed a B-cell phenotype,including CD45R and surface immunoglobulin. Although not by design,applicant shows for the first time that isolated single stromal cellsexpress transcripts that are typically associated with hematopoieticlineage, namely, CD45 and CD19, as well as relevant proto-oncogenes andtranscription factors. These results are strongly supportive of theexistence of a progenitor cell common to bone marrow stromal cells andhematopoietic cells, particularly the bone marrow-derived (B)lymphocytes. Note that the study involves no feeder cells, no embryonicstem cells, no cell lines and no colonies of cells. Contrasting with theexisting literature, the present study embarks on a new path ofinvestigation entailing gene expression analysis of single, primary,normal human stromal cells that suggest a broad capacity formultilineage differentiation. On this model, progenitor cells expressgenes that are characteristic of any of the lineage fates that thesecells are capable of adopting.

Perspective on Pluripotentiality vs. Pluridifferentiation: The presentinvestigation involves isolated single stromal cells, consisting of 10cells from 4 different individuals (FIG. 15). The cell culture systemhas been earlier characterized at light microscopic level,ultrastructural level and by karyotypic analysis, showing no evidence tosuggest the artifacts discussed. Spontaneous cell fusion most likelyinvolves monocytes/macrophages, forming multinucleated giant cells;however applicant observed no expression of myelomonocytic marker genesby isolated single stromal cells (Table 12). Keep in mind that thereadout of in vivo transdifferentiation studies involves localization ofdifferent lineage cells in different tissues or organs; such a situationonly requires fusion between two cell types (one donor cell and onerecipient cell) for the investigators to believe the artifact astransdifferentiation. In contrast, applicant shows here presence ofgenes for a variety of cell-lineages simultaneously within the samecell. The probability of an array of different cell types fusing intoone cell which then masquerading as a pluridifferentiated cell, and thattoo happening with 10 of 10 cells investigated, is in the opinion closeto zero. There has never before been an opportunity to assess the extentof differentiation within these multipotential progenitor cells inmolecular terms at the single-cell level. Theoretically, a) A stem cellcan directly become a terminally differentiated cell, or b) A stem cellcan enter a phase of multilineage differentiation prior to becoming asingle-lineage, mature cell. To the knowledge, this study is the firstsystematic attempt to answer these questions at the single cell level byusing the marrow stromal cells as a model. Although numerousforward-looking reviews exist on the topic of single-cell genomics, onlya rare report is available on the actual application of this technology.Applicant has applied this frontier technology to show that a phase ofmultilineage differentiation indeed exists at least in Dexter-typestromal cells. Pluripotentiality of the bone marrow mesenchymal stromalcells in terms of their ability to become muscle cells, bone cells, fatcells and fibroblasts under select culture conditions has been describedby other investigators. Instead of documenting another example of thephenomenon per se, the results provide an independent validation of thestudies on transdifferentiation by casting light at the molecular basisof cellular plasticity. Finally, to borrow a concept from the clinicalpractice setting, a morphologically “poorly” differentiated neoplasmexpressing hematopoietic markers is classified as a leukemia/lymphomaand treated as such. Similarly, a morphologically “undifferentiated”neoplasm marking for epithelial gene expression is diagnosed as acarcinoma and treated according to the protocols designed for acarcinoma. It is in this sense that applicant uses the term“pluridifferentiated” as opposed to “pluripotential” to characterize theBM stromal cells. Notwithstanding the semantics, applicant shows thatthe pluripotent stromal cells are pluridifferentiated, at least at themolecular level.

Conclusions: The findings of the present study clarify the on-goingcontroversy as to the co-existence of multiple stromal cell types vs.one stromal cell type with co-expression of multiple phenotypes withinthe Dexter system of BM stromal cell cultures. An isolated singlestromal cell from these cultures simultaneously expresses an array ofphenotypes, i.e., osteoblasts, fibroblasts, muscle cells, adipocytes,epithelial cells, endothelial cells, neural cells/glial cells and evenhematopoietic cells, in particular, B-lymphoid progenitors, thusdocumenting its wide differentiation repertoire. The significance of thefindings is three-fold, 1^(st)) They validate the hypothesis that the BMstromal cells express a pluridifferentiated progenitor cell phenotype,providing insight into the molecular basis of cellular plasticity aswell as establishing the utility of single-cell genomics, 2^(nd)) Theyprovide evidence for a common progenitor for mesenchymal progenitors andBM-derived (B) lymphoid progenitors, 3^(rd)) By establishing acomprehensive phenotype of cultured bone marrow stromal cells at singlecell level for the first time, the findings pave the road for ultimateidentification and investigation of these cells in fresh samples ofmarrow, normal as well as diseased, in which they occur at a lowfrequency.

Materials & Methods

Second-Tier Data-Analysis/Data Mining: The microarray data outputted byMAS v.5 (in the form of tab delimited text files) were imported intoGENESPRING software version 4.2.1 (Silicon Genetics, Redwood City,Calif.). Following instructions accompanying GENESPRING, each gene wasnormalized to itself (per-gene normalization) by using the median of thegene's expression values over all the samples of an experimental group(or groups) and dividing each measurement for that gene by thecorresponding median value, assuming that it was at least 0.01. Theratios were then log transformed to base e. No per-sample normalizationwas performed in GENESPRING since it was already accomplished as part ofMAS v.5 analysis. The purpose of the above data transformations,including scaling and normalization, was to remove systematic errorwithin and across conditions or experimental groups prior to furtheranalysis. GENESPRING was used to achieve the following data-analysisobjectives.

a) Filtering for reliably present genes by eliminating the genes withweak expressions that are statistically close to the backgroundestimate. As per the instructions accompanying GENESPRING, random errorwas estimated from control strength or median measurement level usingthe two-component global error model of Rocke-Lorenzato that assumesvariability between replicates, as being similar for all genes showingsimilar measurement level. The formula for the error model of normalizedexpression levels can be written as follows:

S(norm)² =a ² /C ² +b ²

Where, S=standard error of normalized expression data, a & b are the twoerror components, a=an absolute or fixed error component impacting atlower measurement values, b=a relative or proportional error componentimpacting at higher measurement levels, and C=control strength.According to the manufacturer, a curve is fitted for each group ofreplicates, with standard error of normalized data on Y-axis vs. controlstrength on X-axis. At lower end of control strength, the, normalizedstandard error would be high and as the control strength increases, thestandard error would decrease reaching a point where the curve flattensand data become more reliable. Control strength for each condition orsample group at which the above-referred two error components contributeequally, was calculated as follows, for collective MPCs, C=128.68;collective USCs, C=253.52; single-cell MPCs, C=348.32. Each conditionwas filtered for genes expressing signals greater than the respectivecontrol strength, thus eliminating the genes with weak expressions fromeach group. Note 7,196 genes (out of 12,625 gene probes tested) passedthe restriction in case of cMPCs, 7,287 genes in case of cUSCs and 5,937in case of sMPCs. Corresponding gene lists were created.

b) Filtering for genes “present” across replicates in each sample group.GENESPRING's “Add data file restriction” feature was used to prepare therespective lists of genes that were present (or expressed or active) ina least 7 of 8 cUSC samples, 4 of 5 cMPC samples, and 9 of 10 sMPCsamples. Note 5,204 genes (out of 12,625 gene probes tested) passed therestriction in case of cMPCs, 4,763 genes in case of cUSCs, and 3,124genes in case of sMPCs. Corresponding gene lists were created.

c) Exclusion of genes with weak expressions from genes “present” in eachsample group. Respective gene-lists for each sample group from steps (a)and (b), were intersected via Venn diagrams. Note 5,204 genes passed therestriction in case of cMPCs, 4,761 genes in case of cUSCs, and 3,124genes in case of sMPCs, which are almost identical to the numbersobtained as under step (b), except for a difference of 2 genes in caseof cUSCs, thus providing no significant improvement in restricting thedata beyond under step (b). This is a reflection of the high stringencyof the criterion employed under step (b). The 2 genes in case of cUSCsthat passed the restriction under step (b) but failed the combinedrestriction under step (c) did show weak expressions (raw signalsranging, 142-331). Corresponding gene lists were created.

d) Preparation of master stromal cell gene list. Respective gene listsfor the three sample groups from step (c) were intersected via Venndiagrams, resulting in identification of a list of 2,755 genes that areuniformly present or expressed in at least 20 of 23 stromal cell samplesinvestigated. The stromal cell gene list thus arrived at contained genesthat are representative of diverse mesenchymal lineages.Parenthetically, intersecting of gene lists corresponding to the threesample groups from step (b) resulted in a stromal cell gene listconsisting of 2,756 genes, thus differing by I gene from the “official”master stromal cell gene list.

e) Two-way hierarchical clustering of 2755 stromal cell genes based onexpression profiles in 23 stromal cell samples. Only the data that were“cleaned up” of genes with weak expressions as outlined under step (a)were used for hierarchical clustering. This necessitated furtherprocessing of data in MICROSOFT ACCESS prior to analysis by GENESPRING.Note that the data for each individual sample as outputted by MAS v.5contained probe IDs, quantitative and qualitative data, as well as otherinformation such as annotations and are readily recognizable byGENESPRING. In contrast, the gene list, resulting from step (a),contained only probe IDs and could not contain the data associated witheach individual sample and was not recognizable by GENESPRING forinputting as part of an Experiment. Therefore, the microarray data foreach group of individual samples (in EXCEL format) as well as thecorresponding gene list for that group from step (a) (also in EXCELformat) were imported into an ACCESS database. The genes that did notpass the test under step (a) were deleted from the microarray data foreach individual sample by querying and intersecting with the appropriatepost-clean-up gene list. The resulting data files were saved first asEXCEL files, then re-saved as tab delimited text files and then importedinto GENESPRING. Per-gene normalization and log transformation wereapplied as described above. “Gene Tree” and “Experiment Tree” wereconstructed by applying a method similar to that of Eisen et al asimplemented in GENESPRING and by using the stromal cell gene list andthe following parameters: standard correlation as similarity measure; aminimum distance of 0.001; and a separation ratio of 0.5 in case of GeneTree and 1.0 in case of Experiment Tree.

f) Preparation of stromal cell gene lists as relevant to differentcellular phenotypes and/or functions. The gene lists associated withdistinct mesenchymal cell lineages or phenotypes, i.e., osteoblasts,fibroblasts, muscle cells and adipocytes, etc, were prepared using acombination of methods. These include 1) Visually inspecting the entirestromal-cell gene list for relevant key words. 2) Directly searching thestromal cell gene list by using key words of interest via “Advanced FindGenes” feature under Edit menu in GENESPRING and by selecting “SearchOnly Current Gene List”. 3) Intersecting the stromal cell gene list withgene lists of interest from Gene Ontology lists, e.g., list ofoncogenes, via Venn diagrams.

g) Visualization of gene-expression plots. The expression pattern of agene across a given group (or groups) of samples of interest waspictured via Gene Inspector window, utilizing desired display options.

Throughout this application, various publications, are referenced byauthor and year. Full citations for the publications are listed below.The disclosures of these publications in their entireties are herebyincorporated by reference into this application.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology which has been used is intended to bein the nature of words of description rather than of limitation.

The preceding descriptions of the invention are merely illustrative andshould not be considered as limiting the scope of the invention in anyway. From the foregoing description, one of ordinary skill in the artcan easily ascertain the essential characteristics of the instantinvention, and without departing from the spirit and scope thereof, canmake various changes and/or modifications of the inventions to adapt itto various usages and conditions. As such, these changes and/ormodifications are properly, equitably, and intended to be, within thefull range of equivalence of the following claims.

Example 9

The nonhematopoietic stromal cells of the bone marrow are critical forthe development of hematopoietic stem cells into functionally competentblood cells. This study addresses the question of whether bone marrowstromal cell cultures in the Dexter system propagate multiple differentmesenchymal stromal cell types or one stromal cell type that expressesmultiple phenotypes simultaneously. Results show that isolated singlestromal cells simultaneously express transcripts associated withosteoblast, fibroblast, muscle, and adipocyte differentiation.Furthermore, isolated single stromal cells simultaneously expresstranscripts characteristic of epithelial cells, endothelial cells andneural/glial cells. Isolated single stromal cells also expresstranscripts for CD45, CD19, CD10, CD79a, and representativeproto-oncogenes and transcription factors, which are typicallyassociated with normal and neoplastic hematopoietic cells. Thesefindings suggest that the nonhematopoietic mesenchymal cells and thehematopoietic B-lymphocytes have a common progenitor. This is consistentwith the idea that progenitor cells express genes that arecharacteristic of the multiple lineage paths that such cells may becapable of adopting. This study demonstrates the technical feasibilityof transcriptome analysis of individual primary cell-culture grownstromal cells, and supports the concept that bone marrow stromal cellsare relatively homogeneous and show a phenotypic signature of potentialmultilineage differentiation capacity.

As noted above, a recent study from the inventor's laboratory suggestedthe existence of a single unique pluridifferentiated stromal mesenchymalprogenitor cell (MPC) type (B. Seshi et al. Blood Cells Mol. Dis. 26(2000) 234-246). However, the mesenchymal lineage markers usedpreviously are difficult to assess in the same cell. A later study byTremain et al. applied micro-serial analysis of gene expression(micro-SAGE) to determine the “transcriptome” of a single colony-forming“unit-fibroblast” derived from a population of mesenchymal stem cells(MSCs) from Friedenstein system (N. Tremain et al. Stem Cells 19 (2001)408-418). These MSCs (that are relatively less differentiated incomparison to MPCs in Dexter system) also contained transcripts commonto bone, cartilage, muscle, epithelium and neural cells, which supportsthe concept that BM stromal cells express a pluridifferentiatedmesenchymal phenotype. However, the study by Tremain et al. (N. Tremainet al. Stem Cells 19 (2001) 408-418) only analyzed a single colony of BMfibroblasts, CFU-F, consisting of approximately 10,000 cells. Becausesuch a large a colony of cells is not necessarily clonal, it couldpotentially contain multiple discrete singly differentiated mesenchymalcell-types. Another study examined a clonally-derived marrow stromalcell line that expressed the genes representative of all three germlayers (D. Woodbury et al. J. Neurosci. Res. 69 (2002) 908-917),supporting the idea of a pluridifferentiated stromal progenitor cell(Seshi, S. et al. Blood Cells Mol. Dis. 26 (2000) 234-246).

The stromal cell-B cell relationship. Evidence for a progenitor cellcommon to stromal cells and hematopoietic cells has been emerging infragments from isolated reports. Singer et al. in 1984 (J. W. Singer etal. Leuk Res. 8 (1984) 535-545) while investigating bone marrow culturesfrom human patients with clonal myeloproliferative disorders showed thatthe nonhematopoietic stromal cells were derived from the same clonalprogenitors that were involved by the hematopoietic neoplasm, revealedby G6PD marker analysis. Huss et al. in 1995 (R. Huss, et al. Proc.Natl. Acad. Sci. U.S.A. 92 (1995) 748-752) by studying a canine BMstromal cell line showed that the adherent stromal cells had “turned”into nonadherent hematopoietic cells, especially when the latter werecultured in the presence of stem cell factor. Pessina et al. in 1997 (A.Pessina, et al. Exp. Hematol. 25 (1997) 536-541) showed that aparticular murine stromal cell line, upon stimulation with bFGF,expressed a B-cell phenotype, including CD45R and surfaceimmunoglobulin. The present report shows for the first time thatisolated single stromal cells express transcripts that are typicallyassociated with hematopoietic lineage, namely CD45 and CD19, as well asrelevant proto-oncogenes and transcription factors. These resultsstrongly support the existence of a progenitor cell common to bonemarrow stromal cells and hematopoietic cells, particularly the bonemarrow-derived (B) lymphocytes. Even though many of the above genes arenot unique to B cells, the inventor's conclusions are not based onexpression of any one gene. Simultaneous expression of a panel of genes(CD10 +, CD19 +, CD79A +, HLA-Dr +) is indeed unique to pre-B cells. Tothe inventor's knowledge, only pre-B cells and BM stromal cells expressthis composite phenotype.

The experiments presented here use gene expression analysis of isolated,single, primary, normal human bone marrow stromal cells, which are knownto have a broad capacity for multilineage differentiation. The isolatedcells that are the targets of the present analysis are pictured in FIG.15. Progenitor cells express genes that are characteristic of any of thelineage fates that such cells are capable of adopting. Althoughconversion of stromal cells into hematopoietic or B cells has not beenachieved, this work complements the work by earlier investigatorsoutlined above (J. W. Singer et al. Leuk. Res. 8 (1984) 535-545; R. Husset al Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 748-752; A. Pessina et al.Exp. Hematol. 25 (1997) 536-541) and provides new evidence involvinggene expression patterns for possible lineage relationship betweenstromal cells and hematopoietic cells. In addition, this study mayprovide researchers with the tools and information to facilitate asearch for cell culture conditions that permit development of B cellsfrom an isolated single stromal cell.

Pluripotentiality vs. pluridifferentiation. A number of investigatorshave recently shown that hematopoietic stem cells and nonhematopoieticstem cells alike have the capability to transdifferentiate by turningstem cells into variety of tissues revealing their extraordinarypluripotentiality (M. F. Pittenger et al. Science 284 (1999) 143-147; C.R. Bjornson et al. Science 283 (1999) 534-537; M. A. Eglitis et al Proc.Natl. Acad. Sci. U.S. A. 94 (1997) 4080-4085; T. R. Brazelton et alScience 290 (2000) 1775-1779; I. Wilmut et al. [published erratumappears in Nature 1997 Mar. 13; 386(6621):200], Nature 385 (1997)810-813; D. S. Krause et al. Cell 105 (2001) 369-377; Y. Jiang et al.Nature 418 (2002) 41-49). The technical foundations of the studies thatled to the excitement about transdifferentiation or plasticity of stemor progenitor cells have been recently vigorously challenged (N. Teradaet al. Nature 416 (2002) 542-545; Q. L. Ying et al. Nature 416 (2002)545-548; W. A. Wells J. Cell Biol. 157 (2002) 15-18; A. E. Wurmser etal. Nature 416 (2002) 485-487; C. Holden et al. Science 296 (2002)2126-2129; R. Y. Tsai et al. Dev Cell 2 (2002) 707-712; K. Dorshkind NatImmunol 3 (2002) 311-313; S. L. McKinney-Freeman et al. Proc. Natl.Acad. Sci. U.S.A. 99 (2002) 1341-1346). Two technical artifacts thatcould potentially provide misleading results are, a) donor cells canadopt the phenotype of other cells by spontaneous cell fusion, makingthem masquerade as transdifferentiated cells (N. Terada et al. Nature416 (2002) 542-545; Q. L. Ying et al Nature 416 (2002) 545-548) and b)heterogeneity of stem cell types that pre-exist within different tissuesalso can provide misleading results (K. Dorshkind et al. Nat Immunol 3(2002) 311-313; S. L. McKinney-Freeman et al. Proc. Natl. Acad. Sci.U.S.A. 99 (2002) 1341-1346; S. H. Orkin et al. Nat Immunol 3 (2002)323-328). As noted above, the present investigation involves isolatedsingle stromal cells, specifically 10 cells from 4 different individuals(FIG. 15). The cell culture system has been earlier characterized at thelight microscopic level, ultrastructural level and by karyotypicanalysis; these analyses revealed no evidence for spontaneous cellfusion or stem cell heterogeneity (B. Seshi et al. Blood Cells Mol. Dis.26 (2000) 234-246). Spontaneous cell fusion most likely involvesmonocyte/macrophages forming multinucleated giant cells (N. Terada etal. Nature 416 (2002) 542-545); however, it was observed no expressionof myelomonocytic marker genes by isolated single stromal cells (Table17). The interpretation of in vivo transdifferentiation studies involveslocalization of different lineage cells in different tissues or organs;such a situation only requires fusion between two cell types (one donorcell and one recipient cell) for investigators to believe the artifactas transdifferentiation. The probability of an array of different celltypes fusing into one cell, which then masquerade as apluridifferentiated cell, in 10 out of 10 cells studied, is very low.

Although numerous reviews exist on the technology of single-cellgenomics, few studies have applied this technology (D. M. O'Dell et al.Arch. Neurol. 56 (1999) 1453-1456; P. B. Crino et al. Proc. Natl. Acad.Sci. U.S.A. 93 (1996) 14152-14157; J. Cossman J. Histochem. Cytochem. 49(2001) 799-800; J. Eberwine Nat. Neurosci. 4 Suppl (2001) 1155-1156; N.N. Iscove et al. Nat. Biotechnol. 20 (2002) 940-943), and to theinventor's knowledge, this is the first report of successful applicationof the Affymetrix microarray analysis at the single cell level. Theseexperiments were facilitated by the fact that sMPCs are uniquely largecells with abundant, tightly packed cytoplasm and conceivably containrelatively large amount of starting mRNA, as for example compared to alymphocyte.

Investigators have shown that BM stromal cells under select cultureconditions can be turned into bona fide bone cells, muscle cells, fatcells (M. F. Pittenger et al. Science 284 (1999) 143-147; S. Wakitani etal Muscle Nerve 18 (1995) 1417-1426; S. E. Haynesworth et al. Bone 13(1992) 81-88), glial cells (G. C. Kopen et al. Proc. Natl. Acad. Sci.U.S.A. 96 (1999) 10711-10716), and nerve cells (J. Sanchez-Ramos et al.Exp. Neurol. 164 (2000) 247-256; I. B. Black et al. Blood Cells Mol.Dis. 27 (2001) 632-636), demonstrating their pluripotentiality. Bysuggesting a molecular mechanism for stromal cell plasticity, thepresent data support the existence of a common precursor for MPC/neuraland other lineages. These results provide an independent validation ofthe studies on transdifferentiation, such as the extraordinarymultilineage potency of BM-derived stem or progenitor cells, reported byKrause's group (D. S. Krause et al. Cell 105 (2001) 369-377) andVerfaillie's group (Y. Jiang et al. Nature 418 (2002) 41-49). “Lineageburst” characterized by simultaneous activation of diversedifferentiation pathways within the same cell appears to be thesignature profile of the stromal cell, which indicates that a“pluripotent” cell is “pluridifferentiated” at least at the molecularlevel. These results also imply that conversion of a stromal progenitorcell into a terminally differentiated cell (such as bone cell, musclecell, fat cell, fibroblast, etc.) would need to “turn off” the diversecellular pathways that are simultaneously active in a stem or progenitorcell. A recent study showing a clonally-derived BM stromal cell lineexpressed the genes representative of all three germ layers (D. Woodburyet al. J. Neurosci. Res. 69 (2002) 908-917) provides independent supportto the concept of a pluri-differentiated stromal progenitor cell (B.Seshi et al. Blood Cells Mol. Dis. 26 (2000) 234-246). Support alsocomes from the observation that multilineage gene expression precedesunilineage commitment in the hematopoietic system (M. Hu et al. GenesDev. 11 (1997) 774-785).

It is likely that the multipotential cells in the marrow are rare,occurring at an estimated frequency of 1 in 10⁴ nucleated cells (M.Galotto et al. Exp. Hematol. 27 (1999) 1460-1466). However, these cellshave been culture-expanded over 4 weeks. Cultured stromal cellsrepresent the progeny of the stromal cell, and not necessarily thestromal cell itself, for which no in vivo assay as yet exists. Thesuggestion that nonhematopoietic mesenchymal cells and B-lymphocytesshare a common precursor is based on expression of a panel of genes(CD45 +/−, CD34 +/−, CD20 +/−), (CD10 +, CD19 +, CD79A +, HLA-Dr +), andnot expression of CD19 alone. Similar ideas were expressed in a recentpaper (K. Akashi et al. Blood 101 (2003) 383-389) and the accompanyingcommentary (T. Enver Blood 101 (2003) 381). While this paper reportsthat the hematopoietic stem cells of varying potential express the genesassociated with a variety of nonhematopoietic cell types, the presentstudy reports nonhematopoietic stromal progenitor cells which expressthe genes associated with hematopoietic cells, in particular B cells.These two reports raise the question as to how hematopoietic stem cellsand nonhematopoietic stromal cells are related in terms of ontogeny.

Materials and Methods (Example 9)

The present study involved microarray analysis of 23 samples and acorresponding number of chips. The marrow samples were obtained from 4normal healthy adult human subjects, and consisted of mixtures ofunfractionated stromal cells (collective USCs or cUSCs, 8 samples),Percoll gradient-purified mesenchymal progenitor cells (collective MPCsor cMPCs, 5 samples) and single-cell MPCs (sMPCs, 10 samples) obtainedby laser-capture microdissection (LCM) (M. R. Emmert-Buck et al. Science274 (1996) 998-1001), ensuring adequate controls and replicates. Theisolated single stromal cells were selected on the basis of morphology.Wright-Giemsa (or hematoxylin) stained cytospin preparation revealedcharacteristically large nonhematopoietic cells with a relativelyirregular nucleus and cytoplasm compartmentalized into ectoplasm andendoplasm (B. Seshi et al. Blood Cells Mol. Dis. 26 (2000) 234-246).Hematoxylin stain is simpler to use, provides morphologic detailsufficient to allow recognition and isolation of these cells by lasercapture microdissection and does not interfere with downstreammicroarray testing. The photomicrographs of 10 stromal cells that havebeen subjected to microarray testing are shown in FIG. 15. Ascharacterized earlier using immunocytochemical staining (B. Seshi et al.Blood Cells Mol. Dis. 26 (2000) 234-246), the stromal cells targeted formicroarray analysis were CD45-negative cells, thus separating them fromcontaminating hematopoietic cells. To serve as controls and facilitatecomparison, 8 samples of unfractionated stromal cells that were“contaminated” by up to 35% macrophages and 5% hematopoietic cells(cUSC), and 5 samples of Percoll-gradient purified stromal cells, up to95% pure (cMPC) were analyzed side-by-side (B. Seshi et al. Blood CellsMol. Dis. 26 (2000) 234-246). RNA isolated from sMPC samples wassubjected to 2 rounds of amplification using RiboAmp kit (Arcturus, Inc)before in vitro transcription (IVT). In contrast, RNA samples isolatedfrom cUSCs and cMPCs were used without amplification for IVT. Except forthis difference, the steps of microarray testing were standard for all 3types of samples and are outlined as follows: Preparation of totalRNA→generation of cDNA→preparation of ds cDNA→in vitro transcriptioninto cRNA→fragmentation of cRNA→hybridization of target RNA to amicroarray of known genes (Affymetrix U95Av2 oligonucleotide microarray,with 12,625 probe sets)→Signal quantification and first-tier analysisusing the microarray quantification software (Microarray Suite MAS v.5,Affymetrix, Inc). According to the statistical expression analysisalgorithm implemented in MAS v.5, the presence of a gene within a givensample was determined at a detection p-value of <0.05, and was gradedabsent (A), marginal (M) or present/positive (P).

Dexter-Type Bone Marrow Stromal Cell Culture

This study involved bone marrow samples obtained from four healthyadults (3 women and 1 man) ranging in age from 43-50 years. The subjectswere qualified to donate bone marrow for transplantation in a standardclinical BMT setting. Stromal cells were cultured using BM mononuclearcells as the starting cells and following standard protocols as havebeen ongoing in this laboratory, i.e., in presence of hydrocortisone andhorse serum (B. Seshi et al. Blood Cells Mol. Dis. 26 (2000) 234-246; B.Seshi Blood 83 (1994) 2399-2409; S. Gartner et al. Proc. Natl. Acad.Sci. U.S.A. 77 (1980) 4756-4759). The stromal cells representingmesenchymal progenitor cells (MPCs) (˜95% pure), were purified orenriched as described using a discontinuous Percoll gradient afterselective killing of the macrophages in stromal cultures with L-leucinemethyl ester (LME, Sigma) (B. Seshi et al. Blood Cells Mol. Dis. 26(2000) 234-246). Detailed protocols used were published earlier (B.Seshi et al. Blood Cells Mol. Dis. 26 (2000) 234-246). Briefly, the BMmononuclear cells were cultured for 4 weeks, monolayers (FIG. 1) weretrypsinized, and nonhematopoietic cells were purified by Percollgradient (FIG. 2) before they were cytospun in preparation forlaser-capture microdissection (LCM). All samples were treatedidentically. The unfractionated samples contained on average 40%contaminating cells (35% macrophages+5% hematopoietic cells) whereasPercoll gradient-enriched samples contained on average 5% contaminatingcells (macrophages+hematopoietic cells).

Isolation of Individual MPCs Using Laser Capture Microdissection (LCM)

Strict laboratory precautions were observed to ensure preservation ofRNA. All buffers and solutions, e.g. phosphate-buffered saline (PBS) andethanol solutions contained DEPC-treated water. Before microdissectionof individual stromal cells, cytospins of Percoll-purified MPCs wereprepared by attaching dispersed BM stromal cells to uncoated glassslides by low speed (400 rpm) cytocentrifugation using Shandon cytospincentrifuge. The cytospins were fixed in 95% ethanol for 10 min andstained for 30 sec using Hematoxylin QS (Vector, Burlingame, Calif.)followed by washing in DEPC water. The cytospins were then dehydrated inincreasing concentration of ethanol and treated in xylene. This is asingle-step staining method without involving a bluing protocol; itprovided sufficient morphologic detail and did not interfere withdownstream microarray analysis. The MPCs selected on the basis ofmorphology, as visualized on the microscope monitor, were microdissected(M. R. Emmert-Buck et al. Science 274 (1996) 998-1001) using PixCell II(Arcturus, Inc) and captured on CapSure LCM Caps (Arcturus), followed byextraction of RNA (see next).

Microarray Sample Preparation and Testing

Unless mentioned otherwise, sample preparation and microarray testingwere performed according to the protocols outlined in the GENECHIPExpression Analysis Technical Manual (Affymetrix, Inc, Santa Clara,Calif.). RNeasy mini protocol kit (Qiagen, Valencia, Calif.) was usedfor isolation of total RNA from unfractionated stromal cells (USCs) andfrom Percoll-purified MPCs. Superscript II cDNA synthesis kit(Invitrogen) primed with a T7-(dT)₂₄ primer containing a T7 RNApolymerase promoter sequence (Genset Oligos, La Jolla, Calif.) wasemployed to prepare ds cDNA from unamplified RNA samples, using 8-10 μgaliquots of total RNA as the template for first strand cDNA synthesis.The PicoPure RNA Isolation Kit (Arcturus, Mountain View, Calif.) wasemployed for extraction of RNA from LCM-dissected single-cell MPCsamples. RiboAmp RNA amplification kit (Arcturus, Inc) (L. Luo, et al.Nat. Med. 5 (1999) 117-122) was used to amplify RNA from theLCM-dissected single cell samples by performing two rounds ofamplification, terminating the reaction after completion of ds cDNAsynthesis. The entire amplified RNA sample was used as the template forcDNA synthesis. The following steps were identical for both unamplifiedand amplified RNA samples. In vitro transcription (IVT) was performed inthe presence of biotinylated UTP and CTP to produce biotin-labeled cRNA(Bioarray High Yield RNA Transcript labeling Kit, Enzo Diagnostics, Inc,Farmingdale N.Y.), followed by cleaning of the reaction products withRNeasy Mini Kit columns (Qiagen, Valencia, Calif.). The purified,biotin-labeled cRNA samples were then submitted to the Microarray CoreFacility at the University of Florida, Gainesville, where the followingsteps were performed. a) Controlled fragmentation of target cRNA in thepresence of heat and Mg²⁺ b) Hybridization of fragmented cRNA (15 μg)for 16 h at 45° C. to a microarray of known gene probes (U95Av2oligonucleotide microarray, containing ˜12,500 gene probes). c) Washingand staining of the probe array with SAPE (streptavidin phycoerythrin)(Molecular Probes, Inc, Eugene, Oreg.). d) Array scanning with anAgilent argon-ion laser equipped with 488 nm emission and 570 nmdetection wavelengths (GENEARRAY Scanner). e) Backgroundsubtraction/signal quantification and first-tier analysis using thenewest-version of the microarray quantification software, MICROARRAYSuite (MAS v.5, Affymetrix, Inc). That a gene within a given a samplewas absent (A), marginal (M) or present/positive (P) was determined at adetection p-value of <0.05, according to the statistical expressionanalysis algorithm implemented in MAS v.5. First-tier analysis alsoincluded per chip (or per sample) normalization in MAS v.5 by scalingthe trimmed mean signal of a probe-array to a constant target signalvalue of 2,500 to facilitate comparison of the results from differentsamples.

FIGS. 21A-21F shows gene-expression plots of representative precursorB-lymphocyte-associated genes by collective MPCs and single-cell MPCs.Individual samples are represented on X-axis. Signal intensity of atranscript in log scale (normalized across 15 samples) is shown onY-axis. The CD markers that are traditionally associated withhematopoietic cells, CD45 (probe ID 40518_at), CD19 (ID 1116_at) andCD34 (ID (538_at), are expressed by sMPCs. CD45, when present, is moreabundantly detected in single MPCs than in collective MPCs, and isparticularly noticeable by wide range of log scale for CD45. The otherpre-B cell associated markers that are expressed by sMPCs are CD10 (ID1389_at), HLA-Dr (ID 33261_at) and CD79A (ID 34391_at). Samples 1-5,respectively, represent MPC A, MPC B R2, MPC C R2, MPC D R1, MPC D R2.Samples 6-15, respectively, represent SCA1, SCA2, SCA3, SCB1, SCB3,SCC1, SCC3, SCD1, SCD2, and SCD3.

FIGS. 22A-22F show scatter plots using log transformed data and showingsystematic analysis of transcriptome wide random variation. The methodsinvolved in construction of scatter plots are described in the sectionentitled, “Second-tier data-analysis/data mining”. The results arediscussed in the section entitled “Data mining and reproducibility ofoverall procedures”.

Second-Tier Data-Analysis/Data Mining

The microarray data outputted by MAS v.5 (in the form of tab delimitedtext files) were imported into GENESPRING software version 4.2.1(Silicon Genetics, Redwood City, Calif.).

Following instructions accompanying GENESPRING, each gene was normalizedto itself (per-gene normalization) by using the median of the gene'sexpression values over all the samples of an experimental group (orgroups) and dividing each measurement for that gene by the correspondingmedian value, assuming that it was at least 0.01. The ratios were thenlog transformed to base e. No per-sample normalization was performed inGENESPRING because it was already done as part of MAS v.5 analysis. Thepurpose of the above data transformations, including scaling andnormalization, was to remove systematic error within and acrossconditions or experimental groups. GENESPRING was used to achieve thefollowing data-analysis objectives. a) Filtering for reliably presentgenes by eliminating the genes showing weak expressions statisticallyclose to the background estimate. As per the instructions accompanyingGENESPRING, random error was estimated from control strength or medianmeasurement level using the two-component global error model ofRocke-Lorenzato that assumes variability between replicates as beingsimilar for all genes showing similar measurement levels [49]. Theformula for the error model of normalized expression levels may bewritten as follows:

S(norm)² =a ² /C ² +b ²

Where, S=standard error of normalized expression data, a & b are the twoerror components, a=an absolute or fixed error component impacting atlower measurement values, b=a relative or proportional error componentimpacting at higher measurement levels, and C=control strength.According to the manufacturer, a curve is fitted for each group ofreplicates, with standard error of normalized data on Y-axis vs. controlstrength on X-axis. At lower end of control strength, the normalizedstandard error would be high and as the control strength increases, thestandard error would decrease reaching a point where the curve flattensand data become more reliable. Control strength for each condition orsample group, where C=a/b, at which the two error components contributeequally, was calculated as follows, for collective MPCs, C=128.68;collective USCs, C=253.52; single-cell MPCs, C=348.32. Each conditionwas filtered for genes expressing signals greater than the respectivecontrol strength, thus eliminating the genes with weak expressions fromeach group. Of 12,625 gene probes tested, 7,196 genes passed therestriction in case of cMPCs, 7,287 genes in case of cUSCs and 5,937 incase of sMPCs. Corresponding gene lists were created. b) Filtering forgenes “present” across replicates in each sample group. GENESPRING's“Add data file restriction” feature was used to prepare the respectivelists of genes that were present (or expressed or active) in at least 7of 8 cUSC samples, 4 of 5 cMPC samples, and 9 of 10 sMPC samples. Of12,625 genes tested, 5,204 genes passed the restriction in case ofcMPCs, 4,763 genes in case of cUSCs, and 3,124 genes in case of sMPCs.Corresponding gene lists were created. c) Exclusion of genes with weakexpressions from genes “present” in each sample group. Respectivegene-lists for each sample group from steps (a) and (b), wereintersected via Venn diagrams. As a result, 5,204 genes passed therestriction in case of cMPCs, 4,761 genes in case of cUSCs, and 3,124genes in case of sMPCs, which are almost identical to the numbersobtained as under step (b), except for a difference of 2 genes in caseof cUSCs, thus providing no significant improvement in restricting thedata beyond under step (b). This is a reflection of the high stringencyof the criterion used under step (b). The 2 genes in case of cUSCs thatpassed the restriction under step (b) but failed the combinedrestriction under step (c) did show weak expressions (raw signalsranging, 142-331). Corresponding gene lists were created. d) Preparationof the master list of stromal cell genes. Respective gene lists for thethree sample groups from step (c) were intersected via Venn diagrams,resulting in identification of a list of 2,755 genes that are uniformlypresent or expressed in at least 20 of 23 stromal cell samplesinvestigated. The stromal cell gene list thus arrived at contained genesthat are representative of diverse mesenchymal lineages.Parenthetically, intersecting of gene lists corresponding to the threesample groups from step (b) resulted in a stromal cell gene listconsisting of 2,756 genes, thus differing by 1 gene from the master listof stromal cell genes. e) Two-way hierarchical clustering of 2,755stromal cell genes based on expression profiles in 23 stromal cellsamples. Only the data that were “cleaned up” of genes with weakexpressions as outlined under step (a) were used for hierarchicalclustering. This necessitated further processing of data in MICROSOFTACCESS before analysis using GENESPRING.

The data for each individual sample as outputted by MAS v.5 containedprobe IDs, quantitative and qualitative data, as well as otherinformation such as annotations and are readily recognizable byGENESPRING. In contrast, the gene list, resulting from step (a),contained only probe IDs and could not contain the data associated witheach individual sample and was not recognizable by GENESPRING forinputting as part of an Experiment. Therefore, the microarray data foreach group of individual samples (in EXCEL format) as well as thecorresponding gene list for that group from step (a) (also in EXCELformat) were imported into an ACCESS database. The genes that did notpass the test under step (a) were deleted from the microarray data foreach individual sample by querying and intersecting with the appropriatepost-clean-up gene list. The resulting data files were saved first asEXCEL files, then re-saved as tab delimited text files and then importedinto GENESPRING as modified experiments. Per-gene normalization and logtransformation were applied as described above. “Gene Tree” and“Experiment Tree” were constructed by applying a method similar to thatof Eisen et al. (M. B. Eisen et al. Proc. Natl. Acad. Sci. U.S.A. 95(1998) 14863-14868) as implemented in GENESPRING and by using thestromal cell gene list and the following parameters: standardcorrelation as similarity measure; a minimum distance of 0.001; and aseparation ratio of 0.5 in case of Gene Tree and 1.0 in case ofExperiment Tree. f) Preparation of stromal cell gene lists as relevantto different cellular phenotypes and/or functions. The gene listsassociated with distinct mesenchymal cell lineages or phenotypes, i.e.,osteoblasts, fibroblasts, muscle cells and adipocytes, etc., wereprepared using a combination of methods. These include 1) Visuallyinspecting the entire stromal-cell gene list for relevant key words. 2)Directly searching the master list of stromal cell genes by using keywords of interest via “Advanced Find Genes” feature under Edit menu inGENESPRING and by selecting “Search Only Current Gene List”. 3)Intersecting the stromal cell gene list with gene lists of interest fromGene Ontology lists, e.g., list of oncogenes, via Venn diagrams. g)Visualization of gene-expression plots. The expression pattern of a geneacross a given group (or groups) of samples of interest was pictured viaGene Inspector window, utilizing desired display options. h) Statisticalanalysis of random variation in expression of the master list of stromalcell genes vs. the complete list of genes tested. The master list ofstromal cell genes with probe IDs from step (d) was imported intoMICROSOFT ACCESS and intersected with the table containing completeAffymetrix primary data sets (Table 24). The resulting file was exportedas EXCEL file consisting of the master list of stromal cell genes withthe associated Affy data (Table 23). The Affy data as outputted by MASv.5 (in the form of EXCEL Tables 23 and 24) were then imported intoARRAYSTAT software, Version 1.0, Rev. 2.0 (Imaging Research Inc, St.Catharines, ON, Canada). The data for each group of samples were logtransformed to base 10, which allowed the software to construct thescatter plots, standard deviation vs. mean (FIGS. 22A-22F). i)Calculation of basic statistics for different sample groups. The meanand SD values presented as part of Tables 22A-D, 23 and 24 werecalculated using MCG ARRAYSTAT Program (Richard A. MeIndoe, URL:http://www.genomics.mcg.edu/niddkbtc/Software.htm). The accuracy of thereported mean and SD values was checked using EXCEL program.

Morphologic and Phenotypic Characterization of Cell Populations inDexter Cultures

BM stromal cell cultures grown under Dexter conditions (i.e., in thepresence of hydrocortisone and horse serum) are generally considered tobe heterogeneous. Earlier published work, however, showed that Dextercultures are not heterogeneous based on light microscopic,ultrastructural, phenotypic and molecular biological characteristics ofthe nonhematopoietic stromal cells isolated from these cultures (B.Seshi et al Blood Cells Mol. Dis. 26 (2000) 234-246). Detailedcharacteristics of constituent cell populations in Dexter cultures werepublished previously (B. Seshi et al. Blood Cells Mol. Dis. 26 (2000)234-246) and show that nonhematopoietic stromal cells (sMPCs) aremorphologically and phenotypically uniform. Their morphologiccharacteristics are summarized as follows: The sMPCs are large cellswith a relatively large irregular nucleus and abundant cytoplasm that isuniquely compartmentalized into ectoplasm and endoplasm. Macrophages arelarge cells as well, however they have a very small round bullet-likenucleus and foamy cytoplasm. In contrast, hematopoietic cells are smallcells with minimal amount of cytoplasm. Earlier study used Wright-Giemsastain. Comparable data are presented here using hematoxylin staining(FIG. 15) before laser-capture microdissection (LCM). To furthercharacterize these cells, a Percoll-gradient technique was devised forenrichment of nonhematopoietic stromal cells (FIGS. 1 and 2, underDetailed Materials & Methods). While the unfractionated samplescontained on average 40% contaminating cells (35% macrophages+5%hematopoietic cells), the Percoll gradient-enriched samples contained onaverage 5% contamination (macrophages+hematopoietic cells).

Assessment of RNA Amplification Method

The single-cell microarray data were reviewed for reproducibility andvalidity. Two important statistics, reflecting on the reliability of theRNA amplification step, were evaluated. a) 3′:5′ ratios of housekeepingcontrol genes: As shown in Table 5, these ratios were close to I in thestandard unamplified samples, but were increased in the amplifiedsamples. This may reflect preferential amplification toward 3′ end sinceamplification may not proceed all the way to the 5′ end. Alternatively,it may reflect sample degradation. The 3′:5′ ratios were highly variableacross single cell MPC samples in the case of ACTB (beta actin), butwere relatively close in the case of GAPD (glyceraldehyde 3-phosphatedehydrogenase), suggesting that sample degradation did not occur.Furthermore, both GAPD and ACTB gene probes used as part of the standardgene probe set yielded relatively stable signals across replicates ineach sample type, providing further evidence of intactness of RNAsamples targeted for microarray analysis. b) Number of genes present ordetected: As outlined in Table 5, the amplified single cell MPC RNAsamples expectedly showed significantly lower numbers of genes comparedto the standard RNA samples (average 34% V is 46% of the genes etched onthe array). The fact that the number of genes present is relativelyconstant across single cell replicate samples is further indication ofthe data reliability. DNA contamination was unlikely because of the RNAamplification method (which involved Oligo dt-based priming, T7 RNApolymerase-based RNA amplification, and DNase treatment of RNA samplesbefore their purification). Of the 67AFFX hybridization and housekeepingpositive control gene probe sets present on the U95A v2 chip, 22 geneswere detected in at least 7 of 8 cUSC samples, 24 genes were detected in4 of 5 cMPC samples and 19 genes were detected in at least in 9 of 10sMPC samples. Thirteen of these genes were present in at least 20 of the23 samples investigated (Table 20). Similarly, stromal derived factors,SDF1, SDF2 and SDFR1 were detected in at least 20 of the 23 samplesstudied (Table 21).

Data Mining and Reproducibility of Overall Procedures

In many cases, microarray analysis is used to identify genesdifferentially expressed in different sample groups, (i.e., treated vs.untreated, or normal vs. diseased). In contrast, the goal in this studyis to identify genes that are relatively uniformly expressed acrossnormal untreated bone marrow stromal cell samples, regardless whetherthey are of single cell type or populations of cells, purified orunpurified. As described under Materials & Methods, GENESPRING has beenused to achieve the following data-analysis objectives: a) Filtering forgenes reliably detected in each sample group by eliminating the genesshowing weak expression statistically close to the background estimate.b) Filtering for genes that are positive (present) across replicates ineach sample group. c) Exclusion of genes with weak expression from genespresent in each sample group. d) Preparation of a master list of stromalcell genes by intersecting gene lists from step (c) (FIG. 16). Thesesteps have led to identification of a list of 2,755 genes that aredetected in at least 7 of 8 cUSC samples AND 4 of 5 cMPC samples AND 9of 10 sMPC samples (i.e., in at least 20 of 23 stromal cell samplesinvestigated). The main conclusions of the present report are based onwhat is referred to as “master list of stromal cell genes” that isbroadly representative of all 3 types of stromal cell samplesinvestigated, and not on a gene list that is selective to sMPCs.

Not all of the 2,755 positive probes are non-redundant, as there aremultiple probes for many individual genes on the chip employed. Since itwas not possible to determine the actual number of genes that theyrepresent, probe sets and genes are used interchangeably. The list of2,755 genes in the “master list of stromal cell genes” represents 88% ofthe genes expressed by single cells (3,124); 58% of genes expressed byunfractionated samples (4,761); and 53% of genes expressed byPercoll-enriched samples (5,204). The remaining genes expressed bycollective cell samples are probably due to contaminating cells as wellas to genes whose transcripts failed to be amplified in single cellsamples by the amplification method. As indicated in the precedingsection, the amplified single-cell samples detected only ˜34% of thegenes tested (12,625), as opposed to unamplified cell samples whichdetected about 46% of the genes tested. The “remaining genes” listcontains genes associated with myelomonocytic cells, which is consistentwith contaminating cells. The “remaining genes” list also includes anumber of mesenchymal-associated and other genes that failed to beamplified. As previously indicated, the enriched samples contained only5% contaminating cells as opposed to unfractionated stromal cell sampleswhich contained 40% contamination. In light of the high sensitivity ofmicroarray analysis, 5% contamination is probably still sufficient todetect some genes associated with the contaminating cells. Contaminationis recognized as a confounding factor in the analysis of gene expressionresults involving populations of cells, however single cell expressionprofiling, as used here, is free from this artifact.

Hierarchical clustering analysis was used to construct a bone marrowstromal cell tree for visualizing global gene expression patterns acrossreplicates and conditions. As shown in FIG. 17, stromal cell genes thatare expressed at a relatively low level in amplified samples (sMPCs) areclustered to the left of the gene tree; genes that are more stronglyexpressed in sMPCs are prominently configured in the middle of the genetree; and genes that are expressed approximately at the same level asunamplified samples (cMPCs and cUSCs) are clustered to the right of thegene tree. Most importantly, as evident on the sample or experimenttree, hierarchical clustering segregated the members of each sample typeinto a separate group (cMPC, cUSC and sMPC). Within each sample typecorresponding subject replicates clustered together (with minorexceptions). This suggests a fairly high level of reproducibility withinthe data set.

The data in FIG. 18 show that different transcripts amplify to differentextents. The expression of genes within the stromal cell gene listranges from 0.2 to 6 (on the log scale) in unamplified samples (cMPC andcUSC) and from 0.02 to 36 in amplified samples (sMPC), thus showing muchgreater variability in the amplified samples. The effect of differentialamplification is represented graphically using color-coding. Geneexpression curves are colored (following the linear color bar shown onthe right) according to the gene expression level in a particularsingle-cell sample, SCA1. The genes detected at a low level in thissample (as indicated in blue) are not necessarily expressed at a lowlevel in unamplified samples (as read by the log scale on Y-axis). Infact, a significant number of them are expressed at a high level in theunamplified samples. This finding together with the observation thatamplified samples detected about 34% of genes as opposed to unamplifiedsamples detecting about 46% of genes tested (Table 5), suggests thatsome genes do not amplify at all by the method used, whereas other genesamplify to a sufficient degree to be detectable (shown in blue), whilesome other genes amplify to a degree equal to (in yellow) or surpassing(in red) the amounts in the collective samples. (The curve shown inwhite is the housekeeping gene, GAPD.) The statistical algorithmutilized in the latest version of Microarray Analysis Suite (MAS v.5)determined that a gene within a given sample was positive, regardless ofgrading.

Because different transcripts amplify variably, it is not possible tomake a quantitative comparison across transcripts involving theamplified products. However, this does not preclude the usefulness ofthe amplification method for quantitative comparison of a particulartranscript across amplified single cell samples. In fact, the datapoints of a given expression-curve in FIG. 18 are comparable within theamplified samples, suggesting that expression of a particular gene canbe compared in different samples (i.e., normal vs. disease-associatedMPCs). The reproducibility and the fidelity of linear amplification havebeen characterized previously (R. Raja, R. Salunga, T. Taylor, A.Bennett, A. Firouzi, A. Mennis, X.-J. Ma, D. Sgroi, M. Erlander, S.Kunitake, A microgenomics platform for high-throughput gene expressionanalysis of pure cell populations, Journal of Clinical Ligand Assay (inpress) (2003)). It was observed that the spot intensities betweenreplicate amplified samples showed a correlation of r=0.959 and thatamplified and unamplified gene expression ratios of mouse testis/brainshowed a correlation of r=0.913 (R. Raja, R. Salunga, T. Taylor, A.Bennett, A. Firouzi, A. Mennis, X.-J. Ma, D. Sgroi, M. Erlander, S.Kunitake, A microgenomics platform for high-throughput gene expressionanalysis of pure cell populations, Journal of Clinical Ligand Assay (inpress) (2003)). These findings suggest that quantitative comparison ofdifferential gene expression is possible in cases where some but not allRNA samples are amplified.

Documentation of Statistical Variation in Expression of the Master Listof Stromal Cell Genes vs. the Complete List of Genes Present on the Chip

There are multiple ways in which the genes of interest can be selectedfor further study after microarray testing. As outlined above, thestromal cell genes in the master list were selected on the basis oftheir positive calls in at least 20 out of 23 samples investigated. Byplotting the mean expression levels vs. the standard deviation of thelog transformed data, the statistical relationship between theexpression levels vs. the background variation was determined for themaster list of genes, and for the complete list of genes tested. Asshown in FIGS. 22A-22F, the overall variation in the complete list ofgenes showed a negative trend with decrease in the variation as the meansignal strength increased. This result was observed with all three typesof samples investigated. In contrast, similar plots involving the masterlist of stromal cell genes showed flat curves with the random variationor error being relatively constant, suggesting greater reliability oftheir measurements. Also, majority of genes with weak expression havebeen excluded from the master list, as evident from contrasting the meanexpression levels shown on the horizontal axes for complete list ofgenes vs. master list of genes for all three types of samples. Theseobservations would agree with the fact that the genes within the masterlist were to begin with uniformly present or expressed in at least 20out of 23 samples tested.

Multilineage Gene Expression in Single Stromal Cells

A stromal cell gene list, generated as outlined above, is expected to berepresentative of typical stromal cell gene expression profile. Suchmaster list of genes forms the basis for derivation of all other stromalcell gene lists, organized in accordance with lineage or functionalcategories. As depicted in FIGS. 19 & 20, and outlined in Table 18(A-D), these findings show that isolated single cells simultaneouslyexpress genes associated with diverse mesenchymal cell lineages (namely,osteoblast, muscle, fibroblast and adipocyte), suggesting the existenceof a pluridifferentiated mesenchymal progenitor cell type. Analternative interpretation of these findings is that the sensitiveamplification/microarray approach detects levels of transcripts that arenot physiologically relevant and may therefore detect ‘leaky’transcriptional regulation in these cells. While “leaky” transcriptionalregulation is possible, it is unlikely to be the case with sMPCs becausethe genes that formed the major basis for the foregoing conclusions areactive not only in the amplified samples but also in the unamplifiedsamples, ensuring that the results were not unduly biased by low levelexpression occurring only in the single cell samples.

As evident from the other gene lists (Table 18, E-G), an isolated singlestromal cell simultaneously expresses transcripts for epithelial,endothelial and neural cell types as well, widening its transcriptomicrepertoire. Furthermore, as shown in FIG. 21, and Table 18, H-J andTable 17, an isolated single stromal cell expresses transcripts that aretypical of hematopoietic cells, in particular precursor B cells. Thisresult supports the idea that the MPCs within the Dexter system mightrepresent a form or stage of the progenitor cell that is common tononhematopoietic and hematopoietic cells. That BM stromal cells expressCD10 (CALLA) is not novel since BM stromal cells (A. Keating et al Br.J. Haematol. 55 (1983) 623-628) as well as endometrial stromal cells (V.P. Sumathi et al. J. Clin. Pathol. 55 (2002) 391-392) and normal breastmyoepithelial cells (S. Moritani et al. Mod. Pathol. 15 (2002) 397-405)are known to express CD10. However, the simultaneous expression of CD19,CD79A and immunoglobulin enhancer binding factors E12/E47(proto-oncogene TCF3) by BM stromal cells is an unforeseen finding, andforms the basis for postulating the existence of a common progenitorwith B cell lineage. B-cell progenitors typically display the phenotype,(CD45 +/−, CD34 +/−, CD20 +/−), (CD10 +, CD19 +, CD79A +, HLA-Dr +),which is also displayed by isolated single stromal cells at least at thetranscriptome level (Table 17). Primitive CD34+ B cell precursors(so-called Whitlock-Witte initiating cells (C. A. Whitlock et al Proc.Natl. Acad. Sci. U.S.A. 79 (1982) 3608-3612)) express the human homologof the Drosophila Polycomb group gene, BMI1 that appears to be essentialfor the maintenance and proliferation of hematopoietic stem cells (J.Lessard et al. Blood 91 (1998) 1216-1224; J. Lessard et al. Genes Dev.13 (1999) 2691-2703). As reported here, isolated single stromal cellsalso express BMI1 gene (Table 17 and Table 14).

CD45 positivity by cMPC and cUSC samples is attributable to coexistingor “contaminating” hematopoietic cells in these samples as evidenced byconcurrent positivity for myelomonocytic markers CD13, CD33 and CD14(Table 17). However, a similar explanation cannot be used in the case ofisolated single stromal cells. Despite expression of numerousmyeloid-associated proto-oncogenes and transcription factors, none ofthe typical myelomonocytic markers (e.g., CD13, CD33 and CD14) wasidentified in isolated single stromal cells. Similarly, other than CD4,no typical pan T cell lineage markers (e.g., CD5 and CD7) were detectedin stromal cells. CD3 alpha and beta genes were not part of the genechip used and therefore not tested.

The protein products or transcripts for CD45 and CD19 are most likelypresent in stromal cells at a basal level that is beyond the detectionlimits of conventional techniques, e.g., immunocytochemistry andNorthern blotting. Correlation between transcriptome and proteome isestimated to be 0.48-0.76 (S. Hubbard, Functional genomics andbioinformatics,http://www.bi.umist.ac.uk/users/mjfsjh/OPT-GNO/handout2001.htm (2001)),accounting for the discrepancy in findings by conventional techniquesvs. sensitive amplification/microarray analysis. Conceivably, two roundsof amplification prior to IVT sufficiently increased their transcriptlevels to be detected by microarray analysis. In fact, the CD45 levelswere several-fold lower in cMPC and cUSC compared to CD45 levels insMPC, and CD19 was undetectable in unamplified samples. Note that CD45and CD19 are not isolated examples in this regard, since the inventorhas identified at least 200 other genes that are uniquely present acrosssMPC samples but undetectable in cMPC and cUSC (see red circle, FIG.16). These findings could alternatively be interpreted as evidence oflack of fidelity of the amplification method. However, 200 genesrepresent 1.58% the total genes tested (12,625) and 4.67% of the 4,283genes (on average) detected in the amplified samples. Even assuming thisalternative interpretation is correct, the fidelity of amplificationmeasures over 95%. Only one gene was used, namely CD19, from the list ofgenes selective to sMPCs. Even if CD19 were excluded from consideration,the conclusions would still remain unchanged since they are not based onexpression of any one particular gene but rather on simultaneousexpression of a panel of lineage-associated genes.

Finally, the master list of stromal cell genes contained as many as 66human homologs of Drosophila/homeotic genes. Some of these genes areubiquitously expressed in Drosophila, whereas other genes are known fortheir association with specific cellular pathways. As shown in Table 19,the human homologs of Drosophila genes, representing diverse cellularpathways, are simultaneously active in a stromal cell. This findingrepresents additional evidence supporting the existence of apluridifferentiated mesenchymal progenitor cell type.

The gene lists disclosed herein are sub-lists of the master list ofstromal cell genes consisting of 2,755 genes. Detailedlineage-associated gene lists (Tables 6-12 and Tables 14-16) as well asthe master list of stromal cell genes with the associated Affymetrixprimary data (Table 23). Expression of no one gene defines the phenotypeof a particular cell type. Simultaneous expression of a panel of lineagerelated-genes in single isolated cell may be viewed as the harbinger ofa potential cell type. Representative examples of genes corresponding toeach cell lineage are outlined in this table

The stromal cell gene list contains a number of genes that arepotentially capable of causing endothelial differentiation andvasculogenesis within the marrow microenvironment; however, these genesmay not necessarily be endothelial cell markers. In fact, stromal cellsexpress a gene, EDF1, the expression of which inversely correlates withendothelial cell differentiation within the stromal cells, indicatingthat the endothelial cell pathway is being actively “turned off” inthese cells.

Despite expression of numerous myeloid-associated proto-oncogenes andtranscription factors, none of the typical myelomonocytic markers (e.g.,CD13, CD33 and CD14) was identified in stromal cells.

Despite expression of T-cell leukemia associatedproto-oncogenes/transcription factors, no typical pan T-cell lineagemarkers (e.g., CD5 and CD7), other than CD4 and occasional CD2 and CD3epsilon, were identified in stromal cells. CD3 alpha and beta genes werenot part of the gene chip used and therefore not tested.

As noted previously, Table 17 lists stromal cells showing expression ofgenes typically associated with B-cell progenitors. Genes marked withasterisk (*) met the criteria for inclusion in the master list ofstromal-cell genes. Table 17 also shows that typical myelomonocyticmarkers (e.g., CD13, CD33 and CD14), and typical pan T-cell lineagemarkers (e.g., CD5 and CD7) were not detected in single stromal cells,except for occasional CD2 and CD3 epsilon. The Affymetrix primary datacorresponding to the genes listed in Table 17 can be found in Tables22A-D.

A list of human homologs of Drosophila genes, outlining more detaileddescriptions of possible lineage associations is presented as an EXCELfile (Table 13). The Affymetrix primary data corresponding to the geneslisted in Table 19 and Table 13 can be found in Table 23.

All patents, patent applications, provisional applications, andpublications referred to or cited herein, whether supra or infra, areincorporated by reference in their entirety, including all figures,tables, nucleic acid sequence, amino acid sequences, and claims, to theextent they are not inconsistent with the explicit teachings of thisspecification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

Tables

TABLE 5 Summary of human bone marrow stromal cell samples targeted formicroarray analysis with an outline of the corresponding indicators ofassay quality performance. Unfractionated Percoll gradient-LCM-dissected single stromal cells enriched stromal cell samples(Collective USC, 8 cells (Collective (Single Cell MPC, 10 replicates)MPC, 5 replicates) replicates) Subject A UNFR A MPC A SCA1, SCA2, SCA3Subject B UNFR B, UNFR B MPC B R2 SCB1, R1, UNFR B R2 SCB3 Subject CUNFR C R1, MPC C R2 SCC1, UNFR C R2 SCC3 Subject D UNFR D R1, MPC D R1,SCD1, SCD2, UNFR D R2 MPC D R2 SCD3 Amplification of RNA No No Tworounds before IVT Number of genes Mean: 46.63 Mean: 46.54 Mean: 33.93present (% of 12,625) SD: 5.95 SD: 3.66 SD: 3.94 3′:5′ ratio, GAPD Mean:0.89 Mean: 1.23 Mean: 6.76 M33197 (Probe used as SD: 0.33 SD: 0.53 SD:2.97 part of housekeeping control gene probe set) GAPD signal Mean:130,723 Mean: 164,587 Mean: 43,235 35905_s_at (Probe SD: 36,990 SD:40,204 SD: 14,413 used as part of standard (See FIG. 6B.) (See FIG. 6B.)gene probe set) 3′:5′ ratio, ACTB Mean: 1.44 Mean: 2.29 Mean: 57.92X00351 (Probe used as SD: 0.60 SD: 1.57 SD: 67.82 part of housekeepingcontrol gene probe set) ACTB signal Mean: 86,104 Mean: 100,383 ♦Mean:4,445 32318_s_at (Probe SD: 18,458 SD: 28,427 ♦SD: 884 used as part ofstandard (See FIG. 6B.) (See FIG. 6B.) gene probe set) Footnote to Table5 Replicate samples shown under each sample type as indicated correspondto each subject. The replicates of collective USC and collective MPCrepresent cell-culture or biological replicates of stromal cells grownin parallel flasks (instead of technical replicates). Of 27 samples, 2collective MPC samples and 2 single cell MPC samples failed either atthe test chip stage or produced unusual results in terms of the numberof genes present and/or 3′:5′ ratios and were therefore excluded asoutliers. The remaining 23 samples that were targeted for the datamining analysis are represented in this table. The statistics shown(means and SDs) were based on the number of sample replicates indicatedin the top row of the table except for ACTB-signal for single cell MPCs(noted in the table by ♦), which were based on 9 replicates instead of10.

TABLE 6 Osteoblast/bone cell/bone disorders (Seshi, B) Systematic CommonGenbank Description Phenotype/Function 38112_g_at CSPG2 X15998chondroitin sulfate proteoglycan 2 (versican) 38111_at CSPG2 X15998chondroitin sulfate proteoglycan 2 (versican) 38126_at BGN J04599biglycan 36976_at CDH11 D21255 cadherin 11, type 2, OB-cadherin(osteoblast) 37315_f_at BM036 AI057607 uncharacterized bone marrowprotein BM036 36996_at OS-9 U41635 amplified in osteosarcoma 41202_s_atOS4 AF000152 conserved gene amplified in osteosarcoma 671_at SPARCJ03040 secreted protein, acidic, cysteine-rich (osteonectin) 2087_s_atCDH11 D21254 cadherin 11, type 2, OB-cadherin (osteoblast) 1916_s_atc-fos V01512 Human cellular oncogene c-fos OMIM Notes: c-fos (completesequence). restricted to perichondrial growth regions of thecartilaginous skeleton. 1915_s_at c-fos V01512 Human cellular oncogenec-fos (complete sequence). 1388_g_at VDR J03258 vitamin D(1,25-dihydroxyvitamin D3) ?Osteoporosis, receptor involutional;Rickets, vitamin D-resistant 1451_s_at OSF-2 D13666 osteoblast specificfactor 2 (fasciclin I- like) 32094_at CHST3 AB017915 carbohydrate(chondroitin 6) sulfotransferase 3 32307_s_at COL1A2 V00503 collagen,type I, alpha 2 Ehlers-Danlos syndrome, type VIIA2; Marfan syndrome,atypical; Osteogenesis imperfecta, 3 clinical forms, 166200, 166210;Osteoporosis, idiopathic 32306_g_at COL1A2 J03464 collagen, type I,alpha 2 Ehlers-Danlos syndrome, type VIIA2; Marfan syndrome, atypical;Osteogenesis imperfecta, 3 clinical forms, 166200, 166210; Osteoporosis,idiopathic 32305_at COL1A2 J03464 collagen, type I, alpha 2Ehlers-Danlos syndrome, type VIIA2; Marfan syndrome, atypical;Osteogenesis imperfecta, 3 clinical forms, 166200, 166210; Osteoporosis,idiopathic 34321_i_at GS3786 D87120 predicted osteoblast protein34342_s_at SPP1 AF052124 secreted phosphoprotein 1 (osteopontin, bonesialoprotein I, early T-lymphocyte activation 1) 34763_at CSPG6 AF020043chondroitin sulfate proteoglycan 6 (bamacan) 222_at EXT1 S79639exostoses (multiple) 1 Chondrosarcoma; Exostoses, multiple, type 136822_at WAC U51334 WW domain-containing adapter with a Chondrosarcoma,coiled-coil region extraskeletal myxoid 41202_s_at OS4 AF000152conserved gene amplified in osteosarcoma 40790_at BHLHB2 AB004066 basichelix-loop-helix domain containing, /transcription factor class B, 2;OMIM Notes: Alternative title, DEC1, expressed primarily indifferentiated chondrocytes.

TABLE 7 Muscle/muscle disorders (Seshi, B) Systematic Common GenbankDescription Phenotype/Function 38251_at MLC1SA AI127424 myosin lightchain 1 slow a 38923_at FRG1 L76159 FSHD (Facioscapulohumoral musculardystrophy) region gene 1 37012_at CAPZB U03271 capping protein (actinfilament) muscle Z-line, beta 37279_at GEM U10550 GTP binding proteinoverexpressed /GTPase in skeletal muscle 36791_g_at TPM1 M19267tropomyosin 1 (alpha) Cardiomyopathy, familial hypertrophic, 3 36790_atTPM1 M19267 tropomyosin 1 (alpha) Cardiomyopathy, familial hypertrophic,3 36792_at TPM1 Z24727 tropomyosin 1 (alpha) 36678_at TAGLN2 D21261transgelin 2 36641_at CAPZA2 U03851 capping protein (actin filament)muscle Z-line, alpha 2 36931_at TAGLN M95787 transgelin 37631_at MYO1EU14391 myosin IE 41439_at MYO1B AJ001381 myosin IB 40910_at CAPZA1U56637 capping protein (actin filament) /binds barbed ends of muscleZ-line, alpha 1 actin filaments 41187_at MLC-B U26162 myosin regulatorylight chain 41747_s_at MEF2A U49020 Human myocyte-specific enhancerfactor 2A (MEF2A) gene, last coding exon, and complete cds. 41738_atCALD1 M64110 caldesmon 1 41739_s_at CALD1 M83216 caldesmon 1 39791_atATP2A2 M23114 ATPase, Ca++ transporting, cardiac Darier disease muscle,slow twitch 2 39790_at ATP2A2 M23115 ATPase, Ca++ transporting, cardiacDarier disease muscle, slow twitch 2 39378_at BECN1 U17999 beclin 1(coiled-coil, myosin-like BCL2 interacting protein) 40488_at DMD M18533dystrophin (muscular dystrophy, Becker muscular Duchenne and Beckertypes) dystrophy; Cardiomyopathy, dilated, X-linked; Duchenne musculardystrophy 40438_at PPP1R12A D87930 protein (myosin) phosphatase 1, OMIMNotes: Regulates regulatory (inhibitor) subunit 12A the interaction ofactin and myosin downstream of the guanosine triphosphatase Rho.32838_at smooth S67247 Homo sapiens cDNA: FLJ23324 fis, muscle cloneHEP12482, highly similar to myosin heavy HUMMYOHCB Human nonmuscle chainisoform myosin heavy chain-B (MYH10) SMemb mRNA 32755_at ACTA2 X13839actin, alpha 2, smooth muscle, aorta 33994_g_at MLC M22919 Humannonmuscle/smooth muscle alkali myosin light chain gene, complete cds.33447_at MLCB X54304 myosin, light polypeptide, regulatory,non-sarcomeric (20 kD) 32313_at TPM2 M12125 tropomyosin 2 (beta) OMIMNotes: Fibroblast and muscle isoforms result from alternative splicingon exons 6 and 9. 35362_at MYO10 AB018342 myosin X 34306_at MBNLAB007888 muscleblind-like (Drosophila) 36989_at DAG1 L19711 dystroglycan1 (dystrophin- associated glycoprotein 1) 40022_at FCMD AB008226Fukuyama type congenital muscular ?Walker-Warburg dystrophy (fukutin)syndrome; Muscular dystrophy, Fukuyama congenital 39031_at COX7A1AA152406 cytochrome c oxidase subunit VIIa polypeptide 1 (muscle)35729_at MYO1D AB018270 myosin ID 32378_at PKM2 M26252 pyruvate kinase,muscle 40375_at EGR3 X63741 early growth response 3; OMIM Notes:Expressed in deveolping muscle spindles. 1637_at MAPKAPK3 U09578mitogen-activated protein kinase- activated protein kinase 3; OMIMNotes: Expressed especially high in heart and skeletal muscle.40399_r_at MEOX2 A1743406 mesenchyme homeobox 2 (growth arrest-specifichomeobox). OMIM Notes: Important regulator of myogenesis. 39565_atBMPR1A Z22535 bone morphogenetic protein Polyposis, juvenile receptor,type IA. OMIM Notes: intestinal Alternative title, activin receptor-likekinase 3; ALK3. Expressed almost exclusively in skeletal muscle withweak expression in heart and placenta. 41449_at SGCE AJ000534sarcoglycan, epsilon Dystonia, myoclonic

TABLE 8 Fibroblast (Seshi, B) Systematic Common Genbank DescriptionPhenotype/Function 39333_at COL4A1 M26576 Human alpha-1 collagen type IVgene, exon 52. 37037_at P4HA1 M24486 procollagen-proline, 2-oxoglutarate4- dioxygenase (proline 4-hydroxylase), alpha polypeptide I 36666_atP4HB M22806 precursor; Human prolyl 4- hydroxylase beta-subunit anddisulfide isomerase (P4HB) gene, exon 11, clones 6B-(1,3,5,6).41504_s_at MAF AF055376 v-maf musculoaponeurotic fibrosarcoma oncogenehomolog (avian) 39757_at SDC2 J04621 syndecan 2 (heparan sulfateproteoglycan 1, cell surface- associated, fibroglycan) 39945_at FAPU09278 fibroblast activation protein, alpha OMIM Notes: Expressed infetal normal mesenchymal tissues and stromal fibroblasts within commontypes of epithelial tumors. 32835_at MAFF AA725102 v-mafmusculoaponeurotic fibrosarcoma oncogene homolog F (avian) 32535_at FBN1X63556 fibrillin 1 (Marfan syndrome) Ectopia lentis, familial; Marfansyndrome; MASS syndrome; Shprintzen- Goldberg syndrome 2057_g_at FGFR1M34641 fibroblast growth factor receptor 1 Jackson-Weiss sydnrome:(fms-related tyrosine kinase 2, Pfeiffer syndrome Pfeiffer syndrome)1380_at FGF7 M60828 fibroblast growth factor 7 OMIM Notes: May play a(keratinocyte growth factor) role in mesenchymal stimulation ofepithelial cell proliferation. 32313_at TPM2 M12125 tropomyosin 2 (beta)OMIM Notes: Fibroblast and muscle isoforms result from alternativesplicing on exons 6 and 9. 31720_s_at FN1 M10905 fibronectin 1 31719_atFN1 X02761 fibronectin 1 35835_at PDL-108 AB019409 periodontal ligamentfibroblast protein 34390_at P4HA2 U90441 procollagen-proline,2-oxoglutarate 4- dioxygenase (proline 4-hydroxylase), alpha polypeptideII

TABLE 9 Adipocyte (Seshi, B, et al) Probe ID Gene Name Genbank IDDescription OMIM Notes 34378_at ADRP X97324 adiposedifferentiation-related mRNA levels are induced protein(adipophilin)/lipid-droplet rapidly and maximally afterbinding/adipocyte-specific triggering adipocyte differentiation.40282_s_at DF M84526 D component of complement High level of expressionin (adipsin) fat. 33337_at DEGS AF002668 degenerative spermatocytehomolog, lipid desaturase (Drosophila) 39673_i_at ECM2 AB011792extracellular matrix protein 2, female organ and adipocyte specific39674_r_at ECM2 AB011792 extracellular matrix protein 2, female organand adipocyte specific 31504_at HDLBP M64098 high density lipoproteinbinding protein (vigilin) 37542_at LHFPL2 D86961 lipoma HMGIC fusionpartner-like 2 36073_at NDN U35139 necdin homolog (mouse)/Prader- Willisyndrome 37122_at PLIN AB005293 Perilipin (Did not meet the criteriaPlays an important role in to be included in stromal cell gene adipocytemetaboloism. Has list because it was positive in 5 of significantsequence 5 cMPC and 9 of 10 sMPCs, but relationship with ADRP. only 6 of8 cUSc instead of 7 of 8 cUSC samples).

TABLE 10 Epithelial cell/carcinoma (Seshi, B) Systematic Common GenbankDescription Phenotype/Function 38590_r_at PTMA M14630 prothymosin, alpha(gene sequence 28) 38589_i_at PTMA M14630 prothymosin, alpha (genesequence 28) 38610_s_at KRT10; KPP X14467 unnamed protein product; Humangene Epidermolytic for acidic (type I) cytokeratin 10. hyperkeratosis37326_at A4 U93305 integral membrane protein; swiss-prot accession:O04901; may play role in cell differentiation in intestinal epithelium36812_at BCAR3 U92715 breast cancer anti-estrogen resistance 3 36953_atMADH4 U44378 MAD, mothers against decapentaplegic Pancreatic cancer;homolog 4 (Drosophila) Polyposis, juvenile intestinal 36852_at N33U42349 Putative prostate cancer tumor suppressor 36851_g_at N33 U4236039 kDa protein; Human N33 protein ?Prostate cancer, form 2 (N33) gene,exon 11 and susceptibility to complete cds. 37762_at EMP1 Y07909epithelial membrane protein 1 /receptor 37731_at EPS15 Z29064 epidermalgrowth factor receptor pathway substrate 15 40856_at SERPINF1; U29953PEDF; Human pigment epithelium- PEDF; EPC-1 derived factor gene,complete cds. 41431_at ICK AB023153 intestinal cell kinase 39363_at BC-2AF042384 putative breast adenocarcinoma marker (32 kD) 39631_at EMP2U52100 epithelial membrane protein 2 39542_at ENC1 AF059611ectodermal-neural cortex (with BTB-like /associates with domain)p110(RB) 40454_at FAT X87241 FAT tumor suppressor homolog 1 (Drosophila)32781_f_at BPAG1 AA058762 bullous pemphigoid antigen 1 (230/240 kD)32780_at BPAG1 AB018271 bullous pemphigoid antigen 1 (230/240 kD)32329_at KRTHB6 X99142 keratin, hair, basic, 6 (monilethrix) Monilethrix34005_at PIGR X73079 polymeric immunoglobulin receptor, /Binds andtransports expressed in glomerular epithelial cells. polymericimmunoglobulin 1846_at LGALS8 L78132 lectin, galactoside-binding,soluble, 8 (galectin 8); OMIM Notes: Expressed in prostate carcinomacells but only rarely in prostatic hypertrophy.

TABLE 11 Endothelial cell (Seshi, B) Systematic Common GenbankDescription Phenotype/Function 32755_at ACTA2 X13839 actin, alpha 2,smooth muscle, aorta 39315_at ANGPT1 D13628 angiopoietin 1 1929_atANGPT1 U83508 angiopoietin 1 /ligand for the TIE2 receptor 40387_at EDG2U80811 endothelial differentiation, lysophosphatidic acid (LPA) G-protein-coupled receptor, 2 40874_at EDF1 AJ005259 endothelialdifferentiation-related factor 1; OMIM Notes: EDF1 level inverselycorrelates with the level of endothelial differentiation. Inhibition ofEDF1 expression promotes endothelial cell differentiation. It ispostulated that EDF1 may function as a bridging molecule thatinterconnects regulatory proteins and the basal transcriptionalmachinery, thus modulating the transcription of the genes involved inendothelial differentiation. 37907_at F8A; DXS522E M34677 FactorVIII-associated gene 1; CpG island protein; Human nested gene proteingene, complete cds. 41433_at VCAM1 M73255 Human vascular cell adhesionmolecule-1 (VCAM1) gene, complete CDS. 36988_at TNFAIP1 M80783 tumornecrosis factor, alpha-induced protein 1 (endothelial); OMIM Notes:Involved in the promary response of the endothelium to TNF. 583_s_atVCAM1 M30257 vascular cell adhesion molecule 1 1953_at VEGF AF024710vascular endothelial growth factor 36100_at VEGF AF022375 vascularendothelial growth factor 37268_at VEGFB U43368 vascular endothelialgrowth factor B 159_at VEGFC U43142 vascular endothelial growth factor C/ligand and activator of the receptor tyrosine kinase Flt4

TABLE 12 Nerve cell/neuroendocrine/neurologic disorders (Seshi, B)Systematic Common Genbank Description Phenotype/Function 37298_atGABARAP AF044671 GABA(A) receptor-associated protein 37692_at DBIAI557240 diazepam binding inhibitor (GABA receptor modulator,acyl-Coenzyme A binding protein) 35767_at GABARAPL2 AI565760 GABA(A)receptor-associated protein-like 2 35785_at GABARAPL1 W28281 GABA(A)receptor-associated protein like 1 38406_f_at PTGDS AI207842prostaglandin D2 synthase (21 kD, brain) 38657_s_at CLTA M20471clathrin, light polypeptide (Lca), brain. specific insertion sequences38653_at PMP22 D11428 peripheral myelin protein 22 Charcot-Marie-Toothdisease with deafness; Charcot-Marie-Tooth neuropathy-1A; Dejerine-Sottas disease; Neuropathy, recurrent, with pressure palsies 38291_atPENK J00123 preproenkephalin (; Human enkephalin gene: exon 3 and3′flank. 39072_at MXI1 L07648 MAX interacting protein 1Neurofibrosarcoma; Prostate cancer, susceptibility to/ transcriptionfactor; forms heterodimers with Max protein 38841_at GDBR1 AF068195putative glialbiastoma cell differentiation-related 38818_at SPTLC1Y08685 serine palmitoyltransferase, long Neuropathy, hereditary chainbase subunit 1 sensory and autonomic, type 1 36990_at UCHL1 X04741ubiquitin carboxyl-terminal esterase Parkinson disease, familial L1(ubiquitin thiolesterase), neuron- specific. OMIM Notes: Highly specificto neurons and to cells of the diffuse neuroendocrine system and theirtumors. 37005_at NBL1 D28124 neuroblastoma, suppression oftumorigenicity 1 37286_at NRCAM AB002341 neuronal cell adhesion molecule36667_at PYGB U47025 phosphorylase, glycogen; brain 36965_at ANK3 U13616ankyrin 3, node of Ranvier (ankyrin /peripheral proteins G) believed toact as membrane-cytoskeleton linker molecules 38040_at SPF30 AF107463splicing factor 30, survival of motor neuron-related 37958_at BCMP1AL049257 brain cell membrane protein 1 41221_at PGAM1 J04173phosphoglycerate mutase 1 (brain) 40936_at CRIM1 AI651806 cysteine-richmotor neuron 1 41091_at FALZ U05237 fetal Alzheimer antigen. OMIM Notes:Abnormally expressed in fetal brain. The corresponding antibody ALZ50recognizes neurofibrillary pathology associated with Alzheimer'sdisease. 41136_s_at APP Y00264 amyloid beta (A4) precursor proteinAlzheimer disease-1, APP- (protease nexin-II, Alzheimer related;Amyloidosis, disease) cerebroarterial, Dutch type; Schizophrenia,chronic 763_at GMFB AB001106 glia maturation factor, beta 641_at PSEN1L76517 presenilin 1 (Alzheimer disease 3) Alzheimer disease, familial,with spastic paraparesis and unusual plaques; Alzheimer disease-339793_at GBAS AF029786 glioblastoma amplified sequence 40023_at BDNFX60201 brain-derived neurotrophic factor 39687_at E46L AI524873 likemouse brain protein E46 39686_g_at E46L AL050282 like mouse brainprotein E46 39542_at ENC1 AF059611 ectodermal-neural cortex (with BTB-/associates with p110(RB). like domain) OMIM Notes: Expressed highest inbrain. 40193_at ENO2 X51956 Human ENO2 gene for neuron specific (gamma)enolase. 40121_at HIP2 U58522 huntingtin interacting protein 2 40467_atSDHD AB006202 succinate dehydrogenase complex, Paragangliomas, familialsubunit D, integral membrane central nervous system; proteinParagangliomas, familial nonchromaffin, 1, with and without deafness;Pheochromocytoma 40281_at NEDD5 D63378 neural precursor cell expressed,developmentally down-regulated 5 32824_at CLN2 AF039704 deficient inlate-infantile neuronal Ceroid-lipofuscinosis, ceroid lipofuscinosis;Homo sapiens neuronal 2, classic late lysosomal pepstatin insensitiveinfantile protease (CLN2) gene, complete cds. 32607_at BASP1 AF039656brain abundant, membrane attached signal protein 1 33817_at D10S102S63912 FBRNP; heterogeneous ribonucleoprotein homolog; This sequencecomes from FIG. 3; D10S102 = FBRNP [human, fetal brain, mRNA, 3043 nt].33942_s_at STXBP1 AF004563 syntaxin binding protein 1 /implicated invesicle trafficking and neurotransmitter release 1659_s_at RHEB2 D78132Ras homolog enriched in brain 2 1695_at NEDD8 D23662 neural precursorcell expressed, developmentally down-regulated 8 2053_at CDH2 M34064cadherin 2, type 1, N-cadherin (neuronal) 216_at PTGDS M98539 Humanprostaglandin D2 synthase gene, exon 7, brain 32102_at SACS AB018273spastic ataxia of Charlevoix- Spastic ataxia, Charlevoix- Saguenay(sacsin) Saguenay type 31896_at NAG AL050281 neuroblastoma-amplifiedprotein 35681_r_at ZFHX1B AB011141 zinc finger homeobox 1b. OMIMHirschsprung disease- Notes: SMAD-interacting protein 1 mentalretardation (SMADIP1) appears to be essential syndrome; Hirschsprung toembryonic neural and neural crest disease-mental retardationdevelopment. syndrome without Hirschsprung disease 35268_at AXOTAL050171 axotrophin 36190_at CDR2 M63256 cerebellar degeneration-relatedprotein (62 kD) 36609_at SLC1A3 D26443 solute carrier family 1 (glialhigh affinity glutamate transporter), member 3 35973_at HYPH AB023163Huntingtin interacting protein H 36142_at SCA1 X79204 spinocerebellarataxia 1 Spinocerebellar ataxia-1 (olivopontocerebellar ataxia 1,autosomal dominant, ataxin 1) 34817_s_at A2LP U70671 ataxin 2 relatedprotein 34777_at ADM D14874 adrenomedullin 34394_at ADNP AB018327activity-dependent neuroprotector 32606_at BASP1 AA135683 brainabundant, membrane attached signal protein 1 38233_at HOMER-3 AF093265Homer, neuronal immediate early gene, 3 36998_s_at SCA2 Y08262spinocerebellar ataxia 2 Spinocerebellar ataxia-2 (olivopontocerebellarataxia 2, autosomal dominant, ataxin 2) 35150_at TNFRSF5 X60592 tumornecrosis factor receptor Immunodeficiency with superfamily, member 5hyper-IgM, type 3 34166_at SLC6A7 S80071 solute carrier family 6(neurotransmitter transporter, L- proline), member 7 34265_at SGNE1Y00757 secretory granule, neuroendocrine protein 1 (7B2 protein) 654_atMXI1 L07648 MAX interacting protein 1 Neurofibrosarcoma; Prostatecancer, susceptibility to/ transcription factor; forms heterodimers withMax protein 37945_at BACH U91316 brain acyl-CoA hydrolase 39685_at E46LAL050282 like mouse brain protein E46 33769_at MPZL1 AF087020 myelinprotein zero-like 1 39356_at NEDD4L AB007899 neural precursor cellexpressed, developmentally down-regulated 4- like 38800_at STMN2 D45352stathmin-like 2; OMIM Notes: Neuronal growth-associated protein SCG10.36933_at NDRG1 D87953 N-myc downstream regulated gene 1 Neuropathy,hereditary motor and sensory, Lom type 40140_at ZFP103 D76444 zincfinger protein 103 homolog (mouse); OMIM Notes: Alternative title, KF1,expressed in normal cerebellum and Alzheimer disease cerebral cortex,but not in normal cerebral cortex. 1452_at LMO4 U24576 LIM domain only 4OMIM Notes: is highly expressed in the cranial neural crest cells,somite, dorsal limb bud mesenchyme, motor neurons, Schwann cellprogenitors, and T- lymphocyte lineage. 1058_at WASF3 S69790 WAS proteinfamily, member 3

TABLE 13 Drosophila and/or homeotic genes (Seshi, B) Systematic CommonGenbank Description Phenotype/Function 38288_at SNAI2 U69196 snailhomolog 2 (Drosophila). OMIM Notes: Neural crest transcription factorSLUG. A zinc fanger protein that plays an important role in thetransition of epithelial to mesenchymal characteristics within theneural crest. 39037_at MLLT2 L13773 myeloid/lymphoid or mixed-lineageleukemia (trithorax homolog, Drosophila); translocated to, 2 39070_atSNL U03057 singed-like (fascin homolog, sea urchin) (Drosophila). OMIMNotes: Positive in dendritic cells of lymph nodes and Reed-Sternbergcells. 39164_at ARIH2 AF099149 ariadne homolog 2 (Drosophila). OMIMNotes: Upregulated during retinoic acid-induced granulocyticdifferentiation of APL cells. 38750_at NOTCH3 U97669 Notch homolog 3(Drosophila). Cerebral (autosomal OMIM Notes: Promotes the dominant)arteriopathy differentiation of astroglia from with subcortical infarctsmultipotent progenitors. and leukoencephalopathy (CADASIL) 38944_atMADH3 U68019 MAD, mothers against OMIM Notes: SMAD3 decapentaplegichomolog 3 signal transduction (Drosophila) important in the regulationof muscle-specific genes. 37693_at NUMB L40393 numb homolog (Drosophila)OMIM Notes: Numb directs neuronal cell fate decisions. 40004_at SIX1X91868 sine oculis homeobox homolog 1 OMIM Notes: Expressed (Drosophila)in adult skeletal muscle, and in multiple tumors including mammarycarcinoma. 39610_at HOXB2 X16665 homeo box B2 OMIM Notes: Essential formotor neuron development. Within the hematopoietic compartment,expressed specifically in erythromegakaryocytic cell lines. 40575_atDLG5 AB011155 discs, large (Drosophila) homolog 5 OMIM Notes: Expressedin prostate gland epithelial cells. 40570_at FOXO1A AF032885 forkheadbox O1A Rhabdomyosarcoma, (rhabdomyosarcoma) alveolar. OMIM Notes:Activates myogenic transcription program. 40127_at PMX1 M95929 pairedmesoderm homeo box 1 OMIM Notes: Expressed in cardiac, skeletal andsmooth muscle tissues. 40454_at FAT X87241 FAT tumor suppressor homolog1 OMIM Notes: Expressed (Drosophila) in many epithelial, someendothelial and smooth muscle cells. 40328_at TWIST X99268 twist homologSaethre-Chotzen (acrocephalosyndactyly 3; Saethre- syndrome. OMIM Notes:Chotzen syndrome) (Drosophila) Required for cranial neural tubemorphogenesis. 33222_at FZD7 AB017365 frizzled homolog 7 (Drosophila)OMIM Notes: Highest expression adult skeletal muscle and fetal kidney.FZD7 dependent PKC signaling controls cell sorting behaviour in themesoderm. 32696_at PBX3 X59841 pre-B-cell leukemia transcription factor3 33337_at DEGS AF002668 degenerative spermatocyte homolog, lipiddesaturase (Drosophila); adipocyte associated. 1857_at MADH7 AF010193MAD, mothers against OMIM Notes: MAD decapentaplegic homolog 7 proteinswere originally (Drosophila) defined in Drosophila as essentialcomponents of the signaling pathways of the TGF-beta receptor family(e.g., TGFBR1). MADH7 and MADH6 as shown by IHC and ISH arepredominantly expressed in vascular endothelium. 1955_s_at MADH6AF035528 MAD, mothers against /inhibitor of BMP signalingdecapentaplegic homolog 6 (Drosophila) 1013_at MADH5 U59913 MAD, mothersagainst OMIM Notes: SMAD5 decapentaplegic homolog 5 plays a criticalrole in the (Drosophila) signaling pathway by which TGF-beta inhibitsthe proliferation of human hematopoietic progenitor cells. 1453_at MADH2U68018 MAD, mothers against decapentaplegic homolog 2 (Drosophila)1433_g_at MADH3 U68019 MAD, mothers against OMIM Notes: SMAD2/decapentaplegic homolog 3 SMAD3 signal (Drosophila) transduction appearsto be important in the regulation of muscle-specific genes. 35681_r_atZFHX1B AB011141 zinc finger homeobox 1b. OMIM Hirschsprung disease-Notes: SMAD-interacting protein 1 mental retardation (SMADIP1) appearsto be essential syndrome; Hirschsprung to embryonic neural and neuraldisease-mental retardation crest development. syndrome withoutHirschsprung disease 35226_at EYA2 U71207 eyes absent homolog 2 OMIMNotes: Expressed (Drosophila) in extensor tendons, and in lens fibersand participates inconnective tissue patterning. 36308_at ZIC1 D76435Zic family member 1 (odd-paired OMIM Notes: Specifically homolog,Drosophila) expressed in nervous tissue and in particular cerebellargranule cells, potential biomarker for cerebellar granule cell lineageand medulloblastoma. 34306_at MBNL AB007888 muscleblind-like(Drosophila) OMIM Notes: Expressed in skeletal muscle myoblasts, also inlymphoblastoid cell lines. 33710_at C3F U72515 putative protein similarto nessy OMIM Notes: Expressed (Drosophila) in fibroblasts andhepatocytes.

TABLE 14 B-cell/B-cell neoplasms (Seshi, B) Systematic Common GenbankDescription Phenotype/Function 41562_at BMI1 L13689 B lymphoma Mo-MLVinsertion region /proto-oncogene (mouse) 37294_at BTG1 X61123 B-celltranslocation gene 1, anti-proliferative 38418_at CCND1 X59798 cyclin D1(PRAD1: parathyroid Centrocytic lymphoma; adenomatosis 1)Leukemia/lymphoma, B- cell, 1; Multiple myeloma; Parathyroidadenomatosis 1 37730_at p100 U22055 EBNA-2 co-activator (100 kD); OMIMNotes: /associates with the EBV EBNA-2 activates transcription ofspecific nuclear protein 2 acidic genes and is essential forEBV-mediated B- domain lymphocyte transformation. 466_at GTF2I U77948general transcription factor II, I; OMIM Notes: Alternative title,BTK-associated protein, 135 kD (BAP135). Bruton's tyrosine kinase (BTK)is essential for B-cell activation and phosphorylates BAP135 in B cells.36875_at IBTK AL050018 inhibitor of Bruton's tyrsoine kinase 38438_atNFKB1 M58603 nuclear factor of kappa light polypeptide gene enhancer inB-cells 1 (p105) 39730_at ABL1 X16416 v-abl Abelson murine leukemiaviral Leukemia, chronic oncogene homolog 1 myeloid 38743_f_at RAF1X06409 v-raf-1 murine leukemia viral oncogene homolog 1 36645_at RELAL19067 v-rel reticuloendotheliosis viral oncogene homolog A, nuclearfactor of kappa light polypeptide gene enhancer in B-cells 3, p65(avian) 41436_at ZNF198 AJ224901 zinc finger protein 198; OMIM Notes:Stem-cell ZNF198 involves T- or B-cell lymphoblastic leukemia/lymphomalymphoma, myeloid hyperplasia, and syndrome eosinophilia and evolvestoward AML. This multilineage involvement suggests the malignanttransformation of primitive hematopoietic stem cell. 40091_at BCL6U00115 B-cell CLL/lymphoma 6 (zinc finger protein Lymphoma, B-cell,Diffuse 51); OMIM Notes: BCL6 is predominantly Large expressed in theB-cell lineage, especially mature B cells (centrocytes andcentroblasts). 32776_at RALB M35416 v-ral simian leukemia viral oncogenehomolog B (ras related; GTP binding protein) 32696_at PBX3 X59841pre-B-cell leukemia transcription factor 3 33791_at DLEU1 Y15227 deletedin lymphocytic leukemia, 1 34005_at PIGR X73079 polymeric immunoglobulinreceptor /Binds and transports polymeric immunoglobulin 1636_g_at ABLU07563 ABL is the cellular homolog proto-oncogene Leukemia, chronic ofAbelson's murine leukemia virus and is myeloid associated with the t9:22chromosomal translocation with the BCR gene in chronic myelogenous andacute lymphoblastic leukemia. 1728_at BMI1 L13689 B lymphoma Mo-MLVinsertion region /proto-oncogene (mouse) 2020_at CCND1 M73554 cyclin D1(PRAD1: parathyroid Centrocytic lymphoma; adenomatosis 1)Leukemia/lymphoma, B- cell, 1; Multiple myeloma; Parathyroidadenomatosis 1 1295_at RELA L19067 v-rel reticuloendotheliosis viraloncogene homolog A, nuclear factor of kappa light polypeptide geneenhancer in B-cells 3, p65 (avian) 1377_at NFKB1 M58603 nuclear factorof kappa light polypeptide gene enhancer in B-cells 1 (p105) 1461_atNFKBIA M69043 nuclear factor of kappa light polypeptide /IkB-likeactivity gene enhancer in B-cells inhibitor, alpha 1389_at MME J03779membrane metallo-endopeptidase (neutral endopeptidase, enkephalinase,CALLA, CD10) 35350_at GALNAC4 AB011170 B cell RAG associated proteinS-6ST 35992_at MSC AF087036 musculin (activated B-cell factor-1, ABF1);/basic helix-loop-helix OMIM Notes: Downstream target of B-celltranscription factor receptor signal transduction pathway. Alsoexpressed in proliferating undifferentiated myeloblasts. 34344_at IKBKAPAF044195 inhibitor of kappa light polypeptide gene Dysautonomia,familial enhancer in B-cells, kinase complex- associated protein34350_at RSN X64838 restin (Reed-Sternberg cell-expressed intermediatefilament-associated protein); Note R-S cell is a form of B-cell.36204_at PTPRF Y00815 protein tyrosine phosphatase, receptor type, F;OMIM Notes: Alternative title, Leukocyte antigen-related tyrosinephosphatase (LAR). Both LAR and LCA (CD45) map to chromosome 1. LCA isprotein-tyrosine phosphatase, receptor-type C, PTPRC, whereas LAR isPTPRF. 34391_at IGBP1 Y08915 immunoglobulin (CD79A) binding protein 1.IGBP1, a marker for early B-cells 1373_at TCF3 M31523 proto-oncogene ortranscription factor 3 Leukemia, acute TCF3 (E2A immunoglobulin enhancerlymphoblastic. A binding factors E12/E47); OMIM Notes: E2A homeobox genemutant mice will have selective failure to contributing the DNA developB cells, all other hematopoietic cell binding domain of the lineagesbeing intact. The block to B cell t(1:19) translocation developmentoccurs before immunoglobulin protein in precursor B-cell D(H)-J(H)rearrangement. ALL. 35150_at TNFRSF5 X60592 tumor necrosis factorreceptor superfamily, Immunodeficiency with member 5; OMIM notes:Alternative title, B- hyper-IgM, type 3 cell associated molecule CD40;expressed on the surface of all mature B cels, most mature B-cellmalignancies and some early B-cell ALL. 38740_at ZFP36L1 X79067 zincfinger protein 36, C3H type-like 1; OMIM Notes: Alternative title,BERG36 (B- cell early response gene encoding a 36 kD protein). 37026_atCOPEB AF001461 core promoter element binding protein; /transcriptionfactor OMIM Notes: Alternative title, B-cell-derived involved in hepaticwound 1, BCD1. The expression of BCD1 was healing limited to twotissues, CD19+ B-cells and testis of normal individuals. B-cellmaturation is associated with BCD1 expression. 38050_at BTF D79986Bcl-2-associated transcription factor 32696_at PBX3 X59841 pre-B-cellleukemia transcription factor 3

TABLE 15 Myeloid cell/myeloid leukemia (Seshi, B) Systematic CommonGenbank Description Phenotype/Function 39037_at MLLT2 L13773myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog,Drosophila); translocated to, 2 37486_f_at MEIS3 U68385 Meis1, myeloidecotropic viral integration site 1 homolog 3 (mouse) 37685_at PICALMU45976 phosphatidylinositol binding clathrin Leukemia, acute assemblyprotein myeloid; Leukemia, acute T-cell lymphoblastic 41220_at MSFAB023208 MLL septin-like fusion; a fusion partner Leukemia, acute geneof MLL myeloid, therapy- related; Ovarian carcinoma 41175_at CBFB L20298core-binding factor, beta subunit Myeloid leukemia, acute, M4Eo subtype943_at RUNX1 D43968 runt-related transcription factor 1 Leukemia, acute(acute myeloid leukemia 1; aml1 myeloid; Platelet oncogene) disorder,familial, with associated myeloid malignancy 39730_at ABL1 X16416 v-ablAbelson murine leukemia viral Leukemia, chronic oncogene homolog 1myeloid 33146_at MCL1 L08246 myeloid cell leukemia sequence 1(BCL2-related) 1636_g_at ABL U07563 ABL is the cellular homolog proto-Leukemia, chronic oncogene of Abelson's murine myeloid leukemia virusand is associated with the t9:22 chromosomal translocation with the BCRgene in chronic myelogenous and acute lymphoblastic leukemia. 277_atMCL1 L08246 myeloid cell leukemia sequence 1 (BCL2-related) 41388_atMEIS2 AF017418 Meis1, myeloid ecotropic viral integration site 1 homolog2 (mouse) 40189_at SET M93651 SET translocation (myeloid leukemia-associated, M2/M4 AML); SET stands for suppressor of variegation,enhancer of zeste and trithorax. 38992_at DEK X64229 DEK oncogene (DNAbinding) Leukemia, acute nonlymphocytic 36941_at AF1Q U16954 ALL1-fusedgene from chromosome Leukemia, acute 1q myelomonocytic

TABLE 16 T cell/NK cell (Seshi, B) Systematic Common Genbank DescriptionPhenotype/Function 37685_at PICALM U45976 phosphatidylinositol bindingLeukemia, acute myeloid; clathrin assembly protein Leukemia, acuteT-cell lymphoblastic 498_at TAX1BP1 U33821 Tax1 (human T-cell leukemiavirus type I) binding protein 1 40822_at NFATC3 L41067 nuclear factor ofactivated T- cells, cytoplasmic, calcineurin- dependent 3 34003_at CD4U47924 major receptor for HIV-1; /T-cell coreceptor; member ofimmunoglobulin involved in antigen supergene family; T cell surfacerecognition; participant in glycoprotein T4 signal transduction pathway32602_at RAP1GDS1 X63465 RAP1, GTP-GDP dissociation Lymphocyticleukemia, stimulator 1 acute T-cell (T-ALL) 35279_at TAX1BP1 U33821 Tax1(human T-cell leukemia virus type I) binding protein 1 34234_f_at NKTRAI688640 natural killer-tumor recognition sequence; OMIM Notes: Theprotein product of the NKTR gene is present on the surface of LGLs andfacilitates their binding to tumor targets. 39426_at TCERG1 AF017789transcription elongation /HIV-1 Tat transcriptional regulator 1 (CA150)coactivator 32602_at RAP1GDS1 X63465 RAP1, GTP-GDP dissociationLymphocytic leukemia, stimulator 1 acute T-cell

TABLE 17 Stromal cells showing expression of genes that are typicallyaffiliated with B-cell progenitors. Gene name Probe ID Genbank ID cUSCcMPC sMPC CD45 40518_at Y00062 Positive in Positive in Positive in 8/8samples 4/5 samples 6/10 samples CD34 538_at S53911 5/8 4/5 4/10 CD191116_at M28170 0/8 0/5 10/10  CD20 619_s_at M27394 0/8 1/5 3/10 CD2238521_at X59350 2/8 0/5 1/10 * CD10 (CALLA) 1389_at J03779 8/8 5/510/10  * TCF3 (E2A) 1373_at M31523 8/8 5/5 9/10 * CD79A (IGBP1) 34391_atY08915 8/8 5/5 9/10 * HLA class II, Dr 37039_at J00194 8/8 5/5 9/10alpha * HLA class II, Dr 33261_at M16941 8/8 5/5 10/10  beta 1 * B2microglobulin 34644_at AB021288 8/8 5/5 10/10  * BMI1 41562_at L136898/8 5/5 10/10  CD2 40738_at M16336 2/8 1/5 2/10 CD3 epsilon 36277_atM23323 4/8 3/5 3/10 CD5 32953_at X04391 0/8 0/5 0/10 CD7 771_s_at D007490/8 0/5 0/10 CD13 39385_at M22324 8/8 5/5 0/10 CD33 36802_at M23197 4/82/5 0/10 CD14 36661_s_at X06882 8/8 3/5 0/10 Footnote to Table 17 Genesmarked with asterisk * met the criteria for inclusion in stromal cellgene list.

TABLE 18 Stromal cell gene lists associated with diverse cellularlineages. Cell lineage Representative examples of associated genes A)Osteoblast (Table 6) Cadherin 11 (type 2, OB-cadherin, osteoblast),osteonectin, osteopontin, osteoblast specific factor 2 (fasciclinI-like), chondroitin sulfate proteoglycan 2 (versican), biglycan,bamacan, collagen, type I, alpha 2 (Osteogenesis imperfecta) B) Muscle(Table 7) Various types of myosin, tropomyosin 1 and 2, transgelin,transgelin 2, caldesmon 1, dystrophin, dystroglycan 1, Fukuyama typecongenital muscular dystrophy (fukutin), ATPase (Ca⁺⁺ transporting,cardiac muscle, slow twitch 2/Darier disease), capping protein (actinfilament) muscle Z-line (alpha 2 and beta) C) Fibroblast (Table 8)Prolyl 4-hydroxylase, fibronectin, fibrillin 1, fibroglycan, alpha-1collagen type IV gene, fibroblast growth factor 7 (keratinocyte growthfactor) and periodontal ligament fibroblast protein D) Adipocyte (Table9) Adipose differentiation-related protein (adipophilin), adipsin, lipiddesaturase, ECM protein 2 (adipocyte specific), vigilin, necdin andperilipin E) Epithelial cell/carcinoma (Table 10) Cytokeratin 10,keratin (hair, basic, 6, monilethrix), epithelial membrane protein 1,epithelial membrane protein 2, bullous pemphigoid antigen 1, milk fatglobule-EGF factor 8 protein (lactadherin), breast epithelialmucin-associated antigen, prothymosin alpha and thymosin beta 4 and beta10 F) Endothelial cell/angiogenesis/ Angiopoietin, VEGF, VCAM1, FactorVIII- vasculogenesis (Table 11 and Footnote 1) associated gene, EDF1(endothelial differentiation-related factor 1) and EDG2 (endothelialdifferentiation, G-protein-coupled receptor, 2) C) Neural cell (Table12) Neuron-specific (gamma) enolase, GABA receptor-associated proteins,NCAM, N- cadherin, presenilin 1, Huntingtin-interacting protein,adrenomedullin, axotrophin, brain- derived neurotrophic factor (BDNF),syntaxin binding protein 1, peripheral myelin protein 22, ankyrin 3(node of Ranvier, ankyrin G), glial maturation factor, beta H) Myeloidcell/myeloid MLLT2 (mixed-lineage leukemia (trithorax leukemia (Table 15and Footnote 2) homolog, Drosophila), CBFB (core-binding factor, betasubunit), ABL proto-oncogene, MCL1 (myeloid cell leukemia sequence 1)and DEK oncogene I) T cell/NK cell/leukemia CD4, TAX1 binding protein 1,natural killer- (Table 16 and Footnote 3) tumor recognition sequence,RAP1, GTP-GDP association stimulator 1 J) B-cell/B-cell neoplasms (Table14) Bruton's tyrosine kinase-associated protein, 135 kD (BAP135),inhibitor of Bruton's tyrosine kinase, pre-B-cell leukemia transcriptionfactor 3 (PBX3), B cell RAG associated protein, cyclin D1, BCL6, TCF 3(E12/E47), CALLA (CD10), CD79A, COPEB (core promoter element bindingprotein, expression limited to CD19+ B cells and testis), proteintyrosine phosphatase, receptor type, F (similar to CD45, which isPTPRC), restin (expressed in Reed-Sternberg cells in Hodgkin's lymphoma,known as a type of B- cell lymphoma)

TABLE 19 Human homologs of Drosophila genes, representing diversecellular pathways, are simultaneously active in a stromal cell.Representative examples of associated genes Cell lineage Gene Briefdescription Neural SNAI2 snail homolog 2 (Drosophila). NOTCH3 Notchhomolog 3 (Drosophila). NUMB numb homolog (Drosophila) HOXB2 homeo boxB2 TWIST twist homolog (acrocephalosyndactyly 3) (Drosophila) ZIC1 Zicfamily member 1 (odd- paired homolog, Drosophila) ZFHX1B zinc fingerhomeobox 1b. Muscle MADH3 MAD, mothers against decapentaplegic homolog 3(Drosophila) SIX1 sine oculis homeobox homolog 1 (Drosophila) FOXO1Aforkhead box O1A (rhabdomyosarcoma) PMX1 paired mesoderm homeo box 1FZD7 frizzled homolog 7 (Drosophila) MBNL muscleblind-like (Drosophila)MEOX2 mesenchyme homeobox 2. Important regulator of myogenesis.Adipocyte DEGS degenerative spermatocyte homolog, lipid desaturase(Drosophila); adipocyte associated. Epithelial DLG5 discs, large(Drosophila) homolog 5 Endothelial MADH7 MAD, mothers againstdecapentaplegic homolog 7 (Drosophila) Fibroblast EYA2 eyes absenthomolog 2 (Drosophila) C3F putative protein similar to nessy(Drosophila) Hematopoietic MLLT2 myeloid/lymphoid or mixed- lineageleukemia (trithorax homolog, Drosophila); translocated to, 2 SNLsinged-like (fascin homolog, sea urchin) (Drosophila). ARIH2 ariadnehomolog 2 (Drosophila). PBX3 pre-B-cell leukemia transcription factor 3MADH5 MAD, mothers against decapentaplegic homolog 5 (Drosophila)

TABLE 20 Affymetrix (hybridization and housekeeping) positive-controlgenes Probe ID Gene Name Genbank ID Brief description AFFX- ACTB X00351actin, beta HSAC07/X00351_3_st AFFX- ACTB X00351 actin, betaHSAC07/X00351_3_at AFFX-BioC-3_at bioA J04423 ORF 1 AFFX-BioB-M_at bioAJ04423 ORF 1 AFFX-BioDn-5_at bioA J04423 ORF 1 AFFX-BioDn-3_at bioAJ04423 ORF 1 AFFX-BioC-5_at bioA J04423 ORF 1 AFFX- GAPD M33197glyceraldehyde-3-phosphate HUMGAPDH/M33197_3_at dehydrogenase AFFX- GAPDM33197 glyceraldehyde-3-phosphate HUMGAPDH/M33197_5_at dehydrogenaseAFFX- STAT1 M97935 signal transducer and activator ofHUMISGF3A/M97935_3_at transcription 1, 91 kD/ Mycobacterial infection,atypical, familial disseminated AFFX-CreX-5_at X03453 pot. ORF1 (aa1-73); ORF2, put. cre protein (aa 1-343); Bacteriophage P1 cre gene forrecombinase protein. AFFX-CreX-3_at X03453 pot. ORF1 (aa 1-73); ORF2,put. cre protein (aa 1-343); Bacteriophage P1 cre gene for recombinaseprotein. AFFX-hum_alu_at U14573 Human Alu-Sq subfamily consensussequence.

TABLE 21 Stromal-derived factor (SDF) genes active in a stromal cellProbe ID Gene name Genbank ID Brief description 40957_at JJAZ1 D63881joined to JAZF1; Endometrial stromal tumors. OMIM Excerpts: JAZF1/JJAZ1fusion protein present in all types of endometrial tumors. 41627_at SDF2D50645 stromal cell-derived factor 2 32666_at SDF1 U19495 stromalcell-derived factor 1; AIDS, resistance to. OMIM Excerpts: SDF1 inhibitsHIV-1 replication. 33834_at SDF1 L36033 stromal cell-derived factor 1.AIDS, resistance to 35747_at SDFR1 AF035287 stromal cell derived factorreceptor 1 Reference to OMIM: Online Mendelian Inheritance in Man, OMIM(TM). McKusick-Nathans Institute for Genetic Medicine, Johns HopkinsUniversity (Baltimore, MD) and National Center for BiotechnologyInformation, National Library of Medicine (Bethesda, MD), 2000. WorldWide Web URL: http://www.ncbi.nlm.nih.gov/omim/

TABLE 22A List of genes from Table 3 with Affy data & group statisticsGene name Probe ID Descriptions CD45 40518_at Cluster Incl. Y00062:Human mRNA for T200 leukocyte common antigen (CD45, LC-A) /cds =(146,3577) /gb = Y00062 /gi = 34275 /ug = Hs.170121 /len = 4597 CD34538_at S53911 /FEATURE = /DEFINITION = S53911 CD34 = glycoproteinexpressed in lymphohematopoietic progenitor cells {alternativelyspliced, truncated form} [human, UT7, mRNA, 2657 nt] CD19 1116_at M28170/FEATURE = /DEFINITION = HUMCSPC Human cell surface protein CD19 (CD19)gene, complete cds CD20 619_s_at M27394 /FEATURE = cds /DEFINITION =HUMB1LYM Human B-lymphocyte cell-surface antigen B1 (CD20) CD22 38521_atCluster Incl. X59350: H. sapiens mRNA for B cell membrane protein CD22/cds = (56,2599) /gb = X59350 /gi = 36090 /ug = Hs.171763 /len = 3250*CD10 1389_at J03779 /FEATURE = mRNA /DEFINITION = HUMCALLA Human commonacute lymphoblastic leukemia (CALLA) antigen (CALLA) mRNA, complete cds*TCF3 (E2A) 1373_at M31523 /FEATURE = /DEFINITION = HUMTFAA Humantranscription factor (E2A) mRNA, complete cds *CD79A 34391_at ClusterIncl. Y08915: H. sapiens mRNA for alpha 4 protein /cds = (8,1027) /gb =Y08915 /gi = 1877201 (IGBP1) /ug = Hs.3631 /len = 1321 *HLA class II,37039_at Cluster Incl. J00194: human hla-dr antigen alpha-chain mrna &ivs fragments /cds = (26,790) /gb = J00194 /gi = 188231 Dr alpha /ug =Hs.76807 /len = 1199 *HLA class II, 33261_at Cluster Incl. M16941: HumanMHC class II HLA-DR7-associated glycoprotein beta-chain mRNA, Dr beta 1complete cds /cds = (23,823) /gb = M16941 /gi = 188257 /ug = Hs.180255/len = 1146 *B2 34644_at Cluster Incl. AB021288: Homo sapiens mRNA forbeta 2-microglobulin, complete cds /cds = (13,372) /gb = AB021288microglobulin /gi = 4038732 /ug = Hs.75415 /len = 925 *BMI1 41562_atCluster Incl. L13689: Human prot-oncogene (BMI-1) mRNA, complete cds/cds = (479,1459) /gb = L13689 /gi = 291872 /ug = Hs.431 /len = 3203 CD240738_at Cluster Incl. M16336: Human T-cell surface antigen CD2 (T11)mRNA, complete cds, clone PB1 /cds = (23,1105) /gi = 180093 /ug =Hs.89476 /len = 1522 CD3 epsilon 36277_at Cluster Incl. M23323: Humanmembrane protein (CD3-epsilon) gene /cds = (59,682) /gb = M23323 /gi =515731 /ug = Hs.3003 /gb = M16336 /len = 1320 CD5 32953_at Cluster Incl.X04391: Human mRNA for lymphocyte glycoprotein T1/Leu-1 /cds = (72,1559)/gb = X04391 /gi = 37186 /ug = Hs.234745 /len = 2320 CD7 771_s_at D00749/FEATURE = cds /DEFINITION = HUMCD7G3 Human T cell surface antigen CD7gene, exon 4 CD13 39385_at Cluster Incl. M22324: Human aminopeptidaseN/CD13 mRNA encoding aminopeptidase N, complete cds /cds = (120,3023)/gb = M22324 /gi = 178535 /ug = Hs.1239 /len = 3477 CD33 36802_atCluster Incl. M23197: Human differentiation antigen (CD33) mRNA,complete cds /cds = (12,1106) /gb = M23197 /gi = 180097 /ug = Hs.83731/len = 1437 CD14 36661_s_at Cluster Incl. X06882: Human gene for CD14differentiation antigen /cds = (105,1232) /gbX06882 /gi = 29736 /ug =Hs.75627 /len = 1356 Genes marked with asterisk (*) met the criteria forinclusion in the master list of stromal-cell genes (Table 23).

TABLE 22B List of genes from Table 3 with Affy data & group statisticsGene * CD10 * TCF3 name CD45 CD34 CD19 CD20 CD22 (CALLA) (E2A)Collective Probe ID 40518_at 538_at 1116_at 619_s_at 38521_at 1389_at1373_at USC UNFRA 1628 1804 1157.7 74.6 1396.2 7030.2 1619.4 SignalUNFRA P A A A A P P Detection UNFRB 6183.6 2590 148.6 154.7 305.6 60092268.3 Signal UNFRB P A A A A P P Detection UNFRBR1 7040.1 1745.9 967.752.7 1213.7 7322.8 1347.9 Signal UNFRBR1 P P A A A P P Detection UNFRBR28639.9 1465.7 429.1 125.3 1362.8 5749.1 1111 Signal UNFRBR2 P P A A A PP Detection UNFRCR1 3037.7 1728.3 750.2 345.1 2305.3 11343.4 1551.6Signal UNFRCR1 P P A A P P P Detection UNFRCR2 4641.7 1174.5 483.6 11682199.6 13407.3 1638.9 Signal UNFRCR2 P A A P P P P Detection UNFRDR11895.2 2528.8 1195.3 344.3 2114.3 13243.7 2003.3 Signal UNFRDR1 P P A AA P P Detection UNFRDR2 1887.8 2363.5 1092.4 413.9 2069.4 16401.9 1398.4Signal UNFRDR2 P P A A A P P Detection Collective MPCA 879.2 1068 833.9285.9 1676.1 9779.7 2566.7 MPC Signal MPCA P A A A A P P DetectionMPCBR2 326.4 1192.2 549.4 75.8 1110.6 8012.2 1498.1 Signal MPCBR2 A P AA A P P Detection MPCCR2 825.1 1208.7 863.7 1129.7 1232.9 7117.2 1616.6Signal MPCCR2 P P M P A P P Detection MPCDR1 2037.3 2331.6 995.6 48.41654.6 11346.6 1141.3 Signal MPCDR1 P P A A A P P Detection MPCDR21931.3 2450.4 1572.7 470.3 1605.8 10547.7 1413.8 Signal MPCDR2 P P A A AP P Detection Single cell SCA1 109.8 797.9 1018.2 320.5 682.4 16639.82760.1 MPC Signal SCA1 A P P A A P P Detection SCA2 143 835.5 1535 558.2821.1 17433.9 4124.5 Signal SCA2 A A P A A P P Detection SCA3 407.1 8162887.7 663.5 1838 17308.4 3431.5 Signal SCA3 A P P A P P P DetectionSCB1 2736.7 985.1 2716.6 1077.9 319.6 10083.6 5323.7 Signal SCB1 P A P PA P P Detection SCB3 767.3 1170.1 2150 963.4 1137.2 16594 2375.6 SignalSCB3 M P P P A P P Detection SCC1 12806.9 846.4 17005.8 1789.8 1235.227757.7 1826.9 Signal SCC1 P A P A A P P Detection SCC3 17750.2 347.318712.6 707.5 1334.9 30040.6 506.1 Signal SCC3 P A P A A P A DetectionSCD1 7421 1960.6 18621.1 184.3 1909.9 42757.8 1527.5 Signal SCD1 P P P AA P P Detection SCD2 6732.5 1613.3 20745.9 707.8 523.5 42181.6 1816.1Signal SCD2 P M P A A P P Detection SCD3 6130.9 819.2 19101.1 847.21370.4 34681.9 2212.7 Signal SCD3 P A P P A P P Detection Gene * CD79A *HLA class * HLA class II, * B2 CD3 name (IGBP1) II, Dr alpha Dr beta 1microglobulin * BMI1 CD2 epsilon Collective Probe ID 34391_at 37039_at33261_at 34644_at 41562_at 40738_at 36277_at USC UNFRA 1226 38062.13806.9 38013.9 5103.1 459.5 1951.2 Signal UNFRA P P P P P A M DetectionUNFRB 1983.7 132286.8 14982.3 30293 5336.3 1169.5 1115.6 Signal UNFRB PP P P P A A Detection UNFRBR1 2033.4 84235.4 25062.7 55572.2 4871.4 575967.4 Signal UNFRBR1 P P P P P A A Detection UNFRBR2 2176 78133.524972.2 48428.8 5217.7 387.1 885.5 Signal UNFRBR2 P P P P P P PDetection UNFRCR1 1557.9 32586.5 4675.3 52157 3429.9 988.7 985.3 SignalUNFRCR1 P P P P P M A Detection UNFRCR2 1892.3 35945.9 4462.9 565604013.6 512.9 996.4 Signal UNFRCR2 P P P P P P P Detection UNFRDR1 2430.251015.8 11076.1 63105.6 4455.3 802.8 1802.9 Signal UNFRDR1 P P P P P A MDetection UNFRDR2 1789.1 46865.7 6802.8 48011.9 5004.1 892.4 1753.6Signal UNFRDR2 P P P P P A P Detection Collective MPCA 2484.7 16338.16685.1 64828.1 6263.5 754.2 918.2 MPC Signal MPCA P P P P P P ADetection MPCBR2 2076.4 7756.9 2627.9 39862.2 4765.5 349 1220.4 SignalMPCBR2 P P P P P A P Detection MPCCR2 2278.7 7708.7 2497.2 59113.34148.6 485.8 1005.6 Signal MPCCR2 P P P P P A A Detection MPCDR1 1690.537099 7326.5 57360.7 3307.1 960.6 1709 Signal MPCDR1 P P P P P A PDetection MPCDR2 1967.7 36139.8 8206 41709 3629 793.8 2143.1 SignalMPCDR2 P P P P P A P Detection Single cell SCA1 874.1 996.2 1845.129462.7 28699.3 667.6 1305.2 MPC Signal SCA1 P P P P P A P DetectionSCA2 1271.2 1941.2 2295.9 21558.1 27214.8 953.1 1203.5 Signal SCA2 P P PP P A M Detection SCA3 1220.2 1192.1 1884.2 43650.9 15236.4 491.8 495.4Signal SCA3 P A P P P A A Detection SCB1 1470.4 4167.4 2385.2 25705.222346 1454.1 763 Signal SCB1 P P P P P A A Detection SCB3 1198.2 1805.32897.4 22892.5 30792.8 776.2 1162.4 Signal SCB3 P P P P P A P DetectionSCC1 2782.8 12868.8 8143.9 33666.2 28528.7 1345.6 2730.5 Signal SCC1 A PP P P P A Detection SCC3 1715.5 13821.3 6245.5 40680.9 30696.4 741.52316.8 Signal SCC3 P P P P P A M Detection SCD1 2138.1 8080.1 5042.543517.2 29022.8 752.8 1730.5 Signal SCD1 P P P P P M A Detection SCD22022.9 8993.4 3599.8 43695 24371.8 807.1 1087.7 Signal SCD2 P P P P P MM Detection SCD3 2561.8 7539.6 3524.7 43333.9 29904.1 736.3 1004.2Signal SCD3 P P P P P P P Detection Genes marked with asterisk * met thecriteria for inclusion in the master list of stromal-cell genes (Table23).

TABLE 22C Gene name CD5 CD7 CD13 CD33 CD14 Probe ID 32953_at 771_s_at39385_at 36802_at 36661_s_a UNFRA 143.2 648.9 16071.7 497.7 6674.2Signal UNFRA A A P A P Detection UNFRB 159.4 836.4 10077.1 1953.137273.3 Signal UNFRB A A P M P Detection UNFRBR1 70.3 859.9 7365.81294.9 22119.4 Signal UNFRBR1 A A P P P Detection UNFRBR2 62.6 432.16525.3 1016.3 19788.3 Signal UNFRBR2 A A P P P Detection CollectiveUNFRCR1 137 938.3 14153 474.5 10012.2 USC Signal UNFRCR1 A A P A PDetection UNFRCR2 157.5 364.9 9686.8 431.7 6424.2 Signal UNFRCR2 A A P MP Detection UNFRDR1 264.7 731.5 7656.6 1417 14154 Signal UNFRDR1 A A P PP Detection UNFRDR2 245.3 1383.6 9577.5 708.2 14135.1 Signal UNFRDR2 A AP P P Detection MPCA 83.7 503.6 7855.1 328.2 4253.8 Signal MPCA A A P AP Detection MPCBR2 103.2 365.9 2099.1 428 262.3 Signal MPCBR2 A A P A ADetection Collective MPCCR2 70.1 464 16492.9 320.6 1291.2 MPC SignalMPCCR2 A A P A A Detection MPCDR1 174.5 823.9 15377.2 669.2 10550 SignalMPCDR1 A A P P P Detection MPCDR2 213.2 600.7 10487.7 890.8 11953 SignalMPCDR2 A A P P P Detection SCA1 96.1 61.1 1611.5 70.8 133.5 Signal SCA1A A A A A Detection SCA2 77.9 606 2433.8 376.6 165.3 Signal SCA2 A A A AA Detection SCA3 78.8 145.1 2391.8 643.5 100.9 Signal SCA3 A A A A ADetection SCB1 157.5 427.6 1100.4 134.2 156 Signal SCB1 A A A A ADetection SCB3 145.3 297.5 1164.7 116.3 179.7 Signal Single cell SCB3 AA A A A MPC Detection SCC1 435.9 281.7 306.4 406 369.1 Signal SCC1 A A AA A Detection SCC3 166.7 622.2 358.3 107.5 146.3 Signal SCC3 A A A A ADetection SCD1 231.3 539.1 1250.4 580.7 115.8 Signal SCD1 A A A A ADetection SCD2 241.4 189.4 1371.8 432.1 194.9 Signal SCD2 A A A A ADetection SCD3 75.6 295.9 1597.5 104.3 501.4 Signal SCD3 A A A A ADetection

TABLE 22D Collective Collective Single cell Collective Collective Singlecell USC MPC MPC USC MPC MPC Gene Name Mean Standard Deviation CD454,369.25 1,199.86 5,500.54 2,680.72 748.751 5,988.78 CD34 1,925.091,650.18 1,019.14 515.294 679.755 460.488 CD19 778.075 963.06 10,449.40388.856 377.621 8,901.83 CD20 334.825 402.02 782.01 363.374 441.342445.444 CD22 1,620.86 1,456.00 1,117.22 682.519 264.292 531.154 *CD10(CALLA) 10,063.43 9,360.68 25,547.93 4,050.99 1,759.27 11,613.00 *TCF3(E2A) 1,617.35 1,647.30 2,590.47 369.25 542.922 1,390.68 *CD79A (IGBP1)1,886.08 2,099.60 1,725.52 371.138 302.356 632.964 *HLA class II, Dr62,391.46 21,008.50 6,140.54 34,143.40 14,681.30 4,823.50 alpha *HLAclass II, Dr 11,980.15 5,468.54 3,786.42 8,890.23 2,707.57 2,083.76 beta1 *B2 microglobulin 48,892.80 52,574.66 34,816.26 10,578.15 11,129.659,252.53 *BMI1 4,678.93 4,422.74 26,681.31 665.245 1,167.87 4,850.87 CD2723.488 668.68 872.57 281.153 246.894 301.695 CD3 epsilon 1,307.241,399.26 1,379.92 445.605 516.627 691.075 CD5 155 128.94 170.65 72.161.941 111.307 CD7 774.45 551.62 346.56 318.835 173.94 195.186 CD1310,139.23 10,462.40 1,358.66 3,353.99 5,858.02 712.465 CD33 974.175527.36 297.2 547.821 247.232 216.123 CD14 16,322.59 5,662.06 206.2910,176.48 5,331.81 127.73

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1. An isolated pluri-differentiated mesenchymal progenitor cell, whereinsaid cell simultaneously expresses a plurality of genes that are markersfor multiple cell lineages, wherein said multiple cell lineages compriseat least four different mesenchymal cell lineages, and wherein each ofsaid markers is specific for a single cell lineage.
 2. The isolated cellof claim 1, wherein said cell is not a cell of a cell line.
 3. Theisolated cell of claim 1, wherein said at least four differentmesenchymal cell lineages comprise adipocyte, osteoblast, fibroblast,and muscle cell.
 4. The isolated cell of claim 1, wherein said markersspecific for a single cell lineage are selected from the groupconsisting of Nile Red, Oil Red O, adipsin, alkaline phosphatase,cadherin-11, chondroitin sulfate, collagen type I, decorin, fibronectin,prolyl-4-hydroxylase, actin, caldesmon, and transgelin.
 5. The isolatedcell of claim 1, wherein said cell simultaneously expresses saidplurality of genes in the presence of hydrocortisone and horse serum. 6.The isolated cell of claim 1, wherein said cell is not a neoplasticcell.
 7. The isolated cell of claim 1, wherein said cell ischromosomally normal, as determined by Geimsa-trypsin-Wrights (GTW)banding.
 8. The isolated cell of claim 1, wherein said cell is a humancell.
 9. The isolated cell of claim 1, wherein said cell is obtaineddirectly from a primary cell culture.
 10. The isolated cell of claim 9,wherein said primary cell culture is a Dexter culture.
 11. The isolatedcell of claim 1, wherein said cell is obtained by providing a cellculture preparation by the Dexter method, treating the cells of the cellculture preparation to obtain a cell suspension, removing macrophagesfrom the cell suspension, fractionating the remaining cells, andcollecting the fraction of cells containing said isolated cell.
 12. Theisolated cell of claim 1, wherein said cell is not immortalized.
 13. Apharmaceutical composition comprising isolated cells of claim 1 and apharmaceutically acceptable carrier.
 14. The pharmaceutical compositionof claim 13, wherein said cells are present in an amount effective fortreating a disease state in a mammal in need thereof.
 15. Thepharmaceutical composition of claim 14, wherein said cells are presentin an amount effective to enhance hematopoietic progenitor cellengraftment in a mammal in need thereof.
 16. The pharmaceuticalcomposition of claim 14, wherein said cells are present in an amounteffective to treat graft-versus-host disease (GvHD) in a mammal about toundergo bone marrow or organ transplantation or suffering from GvHDcaused by bone marrow or organ transplantation.
 17. The pharmaceuticalcomposition of claim 13, wherein said composition further comprisescells other than pluri-differentiated mesenchymal progenitor cells, ortissue, for transplantation.
 18. The pharmaceutical composition of claim17, wherein said tissue comprises bone marrow.
 19. The pharmaceuticalcomposition of claim 17, wherein said tissue comprises an organ.
 20. Thepharmaceutical composition of claim 13, wherein said at least fourdifferent mesenchymal cell lineages comprise adipocyte, osteoblast,fibroblast, and muscle cell.
 21. The pharmaceutical composition of claim13, wherein said markers specific for a single cell lineage are selectedfrom the group consisting of Nile Red, Oil Red O, adipsin, alkalinephosphatase, cadherin-11, chondroitin sulfate, collagen type I, decorin,fibronectin, prolyl-4-hydroxylase, actin, caldesmon, and transgelin. 22.The pharmaceutical composition of claim 13, wherein said cellssimultaneously express said plurality of genes in the presence ofhydrocortisone and horse serum.
 23. The pharmaceutical composition ofclaim 13, wherein said cells are not neoplastic cells.
 24. Thepharmaceutical composition of claim 13, wherein said cells are notimmortalized.
 25. The pharmaceutical composition of claim 13, whereinsaid cells are chromosomally normal, as determined byGeimsa-trypsin-Wrights (GTW) banding.
 26. The pharmaceutical compositionof claim 13, wherein said cells are human cells.
 27. The pharmaceuticalcomposition of claim 13, wherein said cells are obtained directly from aprimary cell culture.
 28. The pharmaceutical composition of claim 27,wherein said primary cell culture is a Dexter culture.
 29. Thepharmaceutical composition of claim 13, wherein said cells are obtainedby providing a cell culture prepared by the Dexter method, treating thecells of the cell culture to obtain a cell suspension, removingmacrophages from the cell suspension, fractionating the remaining cells,and collecting the fraction containing said pluri-differentiatedmesenchymal progenitor cells.
 30. The pharmaceutical composition ofclaim 13, wherein said pharmaceutically acceptable carrier is sterile.31. The pharmaceutical composition of claim 13, wherein saidpharmaceutically acceptable carrier comprises a sterile saline solution.32. A plurality of isolated pluri-differentiated mesenchymal progenitorcells, wherein said plurality of cells are cells that individuallysimultaneously express a plurality of genes that are markers formultiple cell lineages, wherein said multiple cell lineages comprise atleast four different mesenchymal cell lineages, and wherein each of saidmarkers is specific for a single cell lineage.
 33. The plurality ofcells of claim 32, wherein said plurality of cells are not cells of acell line.
 34. The plurality of cells of claim 32, wherein said at leastfour different mesenchymal cell lineages comprise adipocyte, osteoblast,fibroblast, and muscle cell.
 35. A plurality of pluri-differentiatedmesenchymal progenitor cells, wherein said plurality of cells are cellsthat individually simultaneously express a plurality of genes that aremarkers for multiple cell lineages, wherein said multiple cell lineagescomprise at least four different mesenchymal cell lineages, wherein eachof said markers is specific for a single cell lineage, and wherein saidpluri-differentiated mesenchymal progenitor cells have been isolatedfrom hematopoietic cells and macrophages to a purity of at least 95%.36. The plurality of cells of claim 35, wherein said cells are notimmortalized.
 37. The plurality of cells of claim 35, wherein saidplurality of cells are not neoplastic cells.
 38. The plurality of cellsof claim 35, wherein said plurality of cells are chromosomally normal,as determined by Geimsa-trypsin-Wrights (GTW) banding.
 39. The pluralityof cells of claim 35, wherein said plurality of cells are human cells.40. The plurality of cells of claim 35, wherein said cells are obtaineddirectly from a primary cell culture.
 41. The plurality of cells ofclaim 35, wherein said primary cell culture is a Dexter culture.
 42. Theplurality of cells of claim 35, wherein said plurality of cells areobtained by providing a cell culture preparation by the Dexter method,treating the cells of the cell culture preparation to obtain a cellsuspension, removing macrophages from the cell suspension, fractionatingthe remaining cells, and collecting the fraction of cells containingsaid plurality of cells.
 43. The plurality of cells of claim 35, whereinsaid plurality of cells individually simultaneously express saidplurality of genes in the presence of hydrocortisone and horse serum.44. The plurality of cells of claim 35, wherein said markers specificfor a single cell lineage are selected from the group consisting of NileRed, Oil Red O, adipsin, alkaline phosphatase, cadherin-11, chondroitinsulfate, collagen type I, decorin, fibronectin, prolyl-4-hydroxylase,actin, caldesmon, and transgelin.
 45. A method for purifyingpluri-differentiated mesenchymal progenitor cells comprising the stepsof: (a) providing a cell culture preparation by the Dexter method; (b)treating the cells to obtain a cell suspension; (c) removing macrophagesfrom the cell suspension; (d) fractionating the remaining cells; and (e)collecting the fraction of pluri-differentiated mesenchymal progenitorcells, wherein the pluri-differentiated mesenchymal progenitor cellsindividually simultaneously express a plurality of genes that aremarkers for multiple cell lineages, wherein said multiple cell lineagescomprise at least four different mesenchymal cell lineages, and whereineach of said markers is specific for a single cell lineage.
 46. A methodfor enhancing bone marrow engraftment in a mammal in need thereof, saidmethod comprising administering to the mammal (i) isolatedpluri-differentiated mesenchymal progenitor cells of claim 1 and (ii) abone marrow graft, wherein the isolated pluri-differentiated mesenchymalprogenitor cells are administered in an amount effective to promoteengraftment of the bone marrow in the mammal.
 47. The method of claim46, wherein said administering comprises intravenously injecting ordirectly injecting the isolated pluri-differentiated mesenchymalprogenitor cells to the site of intended engraftment.
 48. A diagnosticmethod for screening isolated pluri-differentiated mesenchymalprogenitor cells for abnormalities, comprising isolating RNA from theisolated pluri-differentiated mesenchymal progenitor cells forabnormalities; amplifying the isolated RNA; analyzing the amplified RNAusing nucleic acid array; determining one or more gene expressionpatterns; and comparing the determined one or more gene expressionpatterns to one or more gene expression patterns of normalpluri-differentiated mesenchymal progenitor cells.
 49. The diagnosticmethod of claim 48, wherein said method is used for screening for ahematologic disease or other diseases effecting stromal cells.
 50. Thediagnostic method according to claim 48, wherein said abnormalities arephenotypic abnormalities that can be discerned at a single cell level.51. A method for reducing graft-versus-host disease (GvHD) in a mammalcaused by bone marrow or organ transplantation, said method comprisingadministering to the mammal an effective amount of isolatedpluri-differentiated mesenchymal progenitor cells of claim
 1. 52. Amethod for diagnosing a disease state comprising the steps of: (a)establishing gene expression patterns of normal state bone marrowderived isolated pluri-differentiated mesenchymal progenitor cells; (b)establishing a gene expression pattern for bone marrow derived isolatedpluri-differentiated mesenchymal progenitor cells of different leukemicstates; (c) identifying gene sets that are unique to a given leukemicstate; and (d) comparing a profile of a bone marrow derived isolatedmesenchymal progenitor cell of unknown state to said gene sets.
 53. Amethod for diagnosing a disease state in a patient, the methodcomprising: (a) providing a gene expression profile of a bone marrowderived isolated pluri-differentiated mesenchymal progenitor cell ofunknown state from the patient; and (b) comparing the patient geneexpression profile to at least one reference gene expression profile todiagnose a disease state in the patient, wherein the reference geneexpression profile is a gene expression profile of a bone marrow derivedisolated pluri-differentiated mesenchymal progenitor cell in a leukemicstate or in a normal state.
 54. The method of claim 53, wherein saidcomparing comprises comparing the patient gene expression profile to aplurality of reference gene expression profiles, wherein each of thereference gene expression profiles is associated with a differentleukemic state.
 55. The method of claim 53, wherein each reference geneexpression profile comprises genes differentially expressed in theleukemic state compared to the normal state.
 56. The method of claim 53,wherein the differentially expressed genes comprise at least one classof genes selected from the group consisting of annexins, caspases,cadherins, calmodulins, calmodulin-dependent kinases, cell adhesionmolecules, cathespins, collagens, cytokines, epidermal growth factors,fibroblast growth factors, fibronectins, galectins, growth factors,genes of the IGF system, interleukins, interleukin receptors, integrins,disintegrins, lineage-specific markers, laminins, platelet-derivedgrowth factors, platelet-derived growth factor receptors,interferon-gamma, TNF-alpha, and TGF-beta.
 57. The method of claim 55,wherein the differentially expressed genes comprise TNF-alpha, TGF-beta,and interferon-gamma.
 58. The method of claim 55, wherein each referencegene expression profile comprises expression values of genesdifferentially expressed in the leukemic state compared to the normalstate.
 59. The method of claim 53, wherein the at least one referencegene expression profile is contained within a database.
 60. The methodof claim 53, wherein said comparing is carried out using a computeralgorithm.
 61. The method of claim 53, wherein said method furthercomprises: (c) selecting the reference gene expression profile mostsimilar to the patient gene expression profile, to diagnose the patient.62. The method of claim 53, wherein said method further comprisesisolating the bone marrow derived isolated pluri-differentiatedmesenchymal progenitor cell of unknown state from the patient.
 63. Themethod of claim 53, wherein the at least one reference gene expressionprofile comprises a gene expression profile of a bone marrow derivedisolated pluri-differentiated mesenchymal progenitor cell in a leukemicstate and a gene expression profile of a bone marrow derived isolatedpluri-differentiated mesenchymal progenitor cell in a normal state. 64.The method of claim 53, wherein said method further comprises preparingthe patient gene expression profile.
 65. The method of claim 53, whereinthe at least one reference gene expression profile has been prepared bycluster analysis.
 66. The method of claim 53, wherein said methodfurther comprises: (c) providing a gene expression profile of a bonemarrow derived isolated pluri-differentiated mesenchymal progenitor cellfrom the patient after the patient has undergone a treatment regimen fora leukemic disease state; and (d) comparing the post-treatment patientgene expression profile to the at least one reference gene expressionprofile, to monitor the patient's response to the treatment regimen. 67.The method of claim 53, wherein the leukemic state is a pre-leukemiccondition.
 68. The method of claim 67, wherein the pre-leukemiccondition is myelodysplastic syndrome (MDS).
 69. The method of claim 53,wherein the leukemic state is an overt leukemia.
 70. The method of claim53, wherein the leukemic state is a lymphoma.
 71. The method of claim53, wherein the leukemic state is selected from the group consisting ofacute myeloid leukemia (AML), chronic myeloid leukemia (CML), acutelymphoblastic leukemia (ALL), chronic lymphocyte leukemia (CLL), andmultiple myeloma (MM).
 72. The method of claim 53, wherein said methodfurther comprises: (c) providing a diagnosis of the disease state to thepatient.
 73. The method of claim 53, wherein the bone marrow derivedisolated pluri-differentiated mesenchymal progenitor cell of unknownstate comprises a single cell.
 74. The method of claim 53, wherein thebone marrow derived isolated pluri-differentiated mesenchymal progenitorcell of unknown state comprises a plurality of cells.
 75. The method ofclaim 53, wherein the isolated pluri-differentiated mesenchymalprogenitor cells have been obtained by providing a cell culturepreparation by the Dexter method, treating the cells of the cell culturepreparation to obtain a cell suspension, removing macrophages from thecell suspension, fractionating the remaining cells, and collecting thefraction of cells containing the normal state pluri-differentiatedmesenchymal progenitor cells.
 76. The method of claim 53, wherein theisolated pluri-differentiated mesenchymal progenitor cells individuallyshare the characteristic of simultaneously expressing a plurality ofgenes that are markers for multiple cell lineages, wherein said multiplecell lineages comprise at least four different mesenchymal celllineages, wherein each of said markers is specific for a single celllineage, and wherein said cells are not cells of a cell line.
 77. Themethod of claim 76, wherein the at least four different mesenchymal celllineages comprise adipocyte, osteoblast, fibroblast, and muscle cell.78. The method of claim 76, wherein the markers specific for a singlecell lineage are selected from the group consisting of Nile Red, Oil RedO. adipsin, alkaline phosphatase, cadherin-11, chondroitin sulfate,collagen type I, decorin, fibronectin, prolyl-4-hydroxylase, actin,caldesmon, and transgelin.
 79. A method for identifying therapeutictargets for treatment of hematopoietic function comprising the steps of:(a) determining the median gene expression profile of isolatedpluri-differentiated mesenchymal progenitor cells associated with eachdisease state of interest; (b) identifying gene groups that areup-regulated, down regulated, and common to each disease state; and (c)identifying gene sets that are unique to a given disease state.
 80. Amouse model for investigating MPC function, wherein said mouse has beenadministered pluri-differentiated mesenchymal progenitor cells of atleast about 95% purity.
 81. The mouse model of claim 80, wherein saidmouse is a Severe Combined Immunodeficiency Disease (SCID) mouse. 82.The mouse model of claim 80, wherein said pluri-differentiatedmesenchymal progenitor cells are of at least 99% purity.
 83. The mousemodel of claim 80, wherein said pluri-differentiated mesenchymalprogenitor cells are human cells.
 84. The mouse model of claim 83,wherein human marrow mononuclear cells have been administered to themouse before, during, or after said human pluri-differentiatedmesenchymal progenitor cells are human cells.
 85. The mouse model ofclaim 84, wherein said human marrow mononuclear cells and said humanpluri-differentiated mesenchymal progenitor cells have been administeredto a site selected from the group consisting of spleen, bone marrow,liver, pancreas, lungs, stomach, and paravertebral neuronal ganglia. 86.A gene in an MPC for detecting the presence of cancer or pre-cancer in acell population.
 87. A pharmaceutical for modulating the gene of claim86.